Dual quencher probes

ABSTRACT

The present invention provides nucleic acid probes comprising two quenchers of excited state energy, and methods of their use.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit under 35 USC 119(e) of U.S.Provisional Application No. 62/147,695, filed Apr. 15, 2015, which isincorporated herein by reference in its entirety for all purposes.

FIELD OF THE INVENTION

The invention resides in the field of labeled nucleic acid probes, andmethods of detecting these probes or fragments of the probes. Exemplaryspecies of the invention are nucleic acid oligomers labeled with two ormore quenching moieties capable of quenching the energy emitted by oneor more fluorescent moiety, which is (are) also conjugated to thenucleic acid oligomer. In general, at least one of the quenchingmoieties and one of the fluorescent moieties are bound at locations onthe oligomer that are sufficiently proximate that detectable energytransfer from the fluorophore to the quencher occurs.

BACKGROUND OF THE INVENTION

The predominant probe type in the qPCR market is linear hydrolysisprobes for use in the 5′ nuclease assay. They are traditionally labeledwith two dyes, generally located at the termini of a nucleic acidoligomer, for example, a 5′ fluorescent reporter and 3′ quencher, e.g.,a dark quencher. The 3′ modification can serve the dual purpose ofprohibiting polymerase extension from the probe upon hybridization; andquenching signal from the fluorophore before the probe hybridizes to itstarget sequence.

The quencher and fluorophore may interact through either FörsterResonance Energy Transfer (FRET) and/or static quenching, which it ishypothesized that they contact one another to form a ground-statecomplex that is non-fluorescent (Marras, S. A. E., Kramer, F. R., andTyagi, S. (2002) Nucleic Acids Res. 30, e122; Johansson, M. K. (2006).Methods Mol. Biol. 335, 17-29). This interaction suppresses the signalwhen the probe is free in solution. Upon oligo hybridization, the duplexformed is quite rigid and so increases the effective length compared tosingle-stranded oligo, which is much more flexible. Binding of the probeto the target sequence is therefore sufficient to disrupt theinteraction between dyes positioned at opposite ends of an oligo andrelease fluorescent signal (Parkhurst, K. M., and Parkhurst, L. J.(1995). Biochemistry 34, 285-292).

The signal release upon hybridization can be made permanent byhydrolyzing the oligo between the fluorophore and the quencher. In fact,cleavage is accomplished when Taq polymerase extends from a primer andencounters the probe in its path (FIG. 1). The nuclease activity of thepolymerase cleaves the probe one or several bases from its 5′ end untilthe probe loses binding stability and is displaced from the strand. Thishydrolysis is the basis for the signaling mechanism in TaqMan® probes.

FIG. 1 is an illustration of a 5′ nuclease (TaqMan®) assay. Briefly,after denaturation, the probe and primers anneal to the dissociatedstrands. During elongation the polymerase extends from the primer,encounters the probe, and hydrolyzes nucleotide fragments that aredisplaced from the strand. Cleavage of the probe separates thefluorophore and quencher rendering signal release permanent.

Probe performance can be quantified in a general sense by asignal-to-noise ratio: the final fluorescence following amplificationdivided by the initial fluorescence preceding amplification. The initialfluorescence represents the quenching efficiency, and so to double thisbackground signal reduces the S:N by a factor of two. Quenchingefficiency depends, in part, upon the oligo length. This is because FRETquenching diminishes quite rapidly with increasing separation betweenthe dyes according to a relationship of (1/r){circumflex over ( )}6,where r is the distance through space (Cardullo et al. (1988). Proc.Natl. Acad. Sci. U.S.A. 85, 8790-8794.). Single-stranded oligos arethought to behave as a random coil and so the effective distance is theaverage of many conformations, but the principle remains the same:increasing sequence length diminishes the quenching efficiency,resulting in probes with elevated baseline fluorescence and poorsignal-to-noise values. For this reason, probe designs are typicallylimited to 30 bases or shorter to achieve sufficient quenchingefficiency for the final application.

Oligonucleic acid lengths are selected with consideration not only toquenching efficiency but also their binding stability. A common designguideline is to select probe sequences with a T_(M) of 70° C., elevatedabove that of the primers. Probes must typically be longer than 20 basesto accomplish that T_(M) depending on the base composition, and in theabsence of special modifications to promote hybridization. Sequencedesign is thus a careful compromise between binding stability andquenching efficiency, but that 20-30 base window is inadequate for manydifficult targets. For example, SNP genotyping requires probes shorterthan 20 bases in order to achieve the enhanced specificity needed formismatch discrimination. At such a low T_(M) SNP probes would benonfunctional without the use of chemical moieties to increase theirbinding stability—a Minor Groove Binder (MGB) (Kutyavin et al. (2000).Nucleic Acids Res. 28, 655-661) or the propynyl residues used in BHQplusprobes, among others. These chemical modifications serve to relax thelower limitation, allowing the design of compact sequences beneath 20bases in length.

Many target genes are particularly AT-rich and require longer sequencesto obtain the proper T_(M). The upper limitation on sequence length canbe relaxed by positioning the quencher at an internal location closer tothe fluorescent reporter, rather than at the 3′ terminus. In fact, someof the earliest TaqMan probe designs had the quencher at an internallocation for this reason—to improve their quenching efficiency (Lee etal. (1993) Nucleic Acids Res. 21(16): 3761-3766). However, the 3′position now vacated must still be modified with a blocker such as aterminal phosphate or aliphatic carbon spacer, to prevent the probe frombehaving as a primer and triggering extension.

Probes with an internal quencher do improve the quenching efficiency forsequences longer than 30-bases. Internal quenchers are traditionallypositioned off of a thymidine base to preserve the sugar-phosphatebackbone and presumably minimize any disruption to base-pairing. Forcertain assays, however, this strategy will diminish the magnitude ofsignal release upon amplification. The reasons for this suppression arenot entirely clear, but the outcome effectively reduces the numerator ofthe S:N ratio, compromising probe performance. FIGS. 2A-C showrepresentative results when Black Hole Quencher-1 (BHQ1) is positionedupon an internal T residue, across three different probe lengths.

FIGS. 2A-C. Amplification traces signaled with an end-labeled probe areshown as slashed lines. Amplification traces signaled with an internalT-BHQ1 probe are shown as solid lines. The improvement in quenchingefficiency is most pronounced with longer probe sequences, while theshort 21-base probe has a reduced magnitude of signal release comparedto the end-labeled probe.

Tethering the BHQ1 off a thymidine constrains probe design to thosesequences that have a T toward the 5′ end, since the reactive precursorused to incorporate the quencher during oligo synthesis involves aT-linked phosphoramidite. This base dependence is a significantlimitation, as is the reduced signal release from select assays,particularly those with shorter probe sequences. The design of qPCRassays could be made more facile, and their signal amplification morerobust by:

-   -   1. Improving both the magnitude of signal release and the        quenching efficiency.    -   2. Eliminating any base dependencies that restrict the quencher        position.    -   3. A label orientation that permits design of both short and        long sequences with similar performance improvement.

The present invention provides nucleic acid probes meeting these threeand having other advantages as well.

SUMMARY OF THE INVENTION

The present invention provides nucleic acid probes comprising twoquenchers of excited state energy, and methods of their use.

Other objects, advantages and aspects of the present invention will beapparent from the detailed description below.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is an illustration of a 5′ nuclease (TaqMan®) assay.

FIG. 2A-FIG. 2C show the amplification traces for end-labeled probes(slashed) and internal T-BHQ1 labeled probes (solid) at various probelengths.

FIG. 3 shows the structure of an exemplary dual quencher probe of theinvention.

FIG. 4A-FIG. 4C show the amplification traces for end-labeled probes(slashed) and dual quencher probes (solid) at various probe lengths.

FIG. 5A-FIG. 5F show the (raw and normalized) amplification traces forend-labeled probes (slashed) and dual quencher probes (solid) at variousprobe lengths.

FIG. 6A-FIG. 6B FIG. 6A shows the amplification traces for anend-labeled probe (slashed) and an I-TBHQ1+3-BHQ1 probe (solid). FIG. 6Bshows the amplification traces for an end-labeled probe (slashed) and anI-BHQ1+3-BHQ1 probe (solid).

FIG. 7A-FIG. 7B FIG. 7A shows the amplification traces for anend-labeled probe (slashed), an I-BHQ0+3-BHQ1 probe (solid), and anI-TDAB+3-BHQ1 probe (hollow). FIG. 7B shows the amplification traces foran end-labeled probe (slashed), an I-BHQ0+3-BHQ1 probe (solid), and anI-Aniline+3-BHQ1 probe (hollow).

FIG. 8A-FIG. 8B FIG. 8A shows the amplification traces for anend-labeled probe (slashed), an I-BHQ0+3-BHQ1 probe (solid), and anI-dRBHQ0+3-BHQ1 probe (hollow). FIG. 8B shows the amplification tracesfor an end-labeled probe (slashed), an I-BHQ0+3-BHQ1 probe (solid), andan I-gBHQ0+3-BHQ1 probe (hollow).

FIG. 9A-FIG. 9B FIG. 9A shows the amplification traces for anend-labeled probe (slashed), an I-BHQ0+3-BHQ1 probe (solid), and anI-BHQ0+3-Sp3 probe (hollow).

FIG. 9B shows the amplification traces for an end-labeled probe(slashed), an I-BHQ0+3-BHQ1 probe (solid), and an I-BHQ1+3-BHQ0 probe(hollow).

FIG. 10 shows the amplification traces for an end-labeled probe(slashed), and various I-BHQ0+3-BHQ1 probes.

FIG. 11A-FIG. 11L show the melting curves for end-labeled probes(slashed), I-BHQ0+3-BHQ1 probes (solid), and probes without complement(hollow), respectively, for different probe sequences.

DETAILED DESCRIPTION OF THE INVENTION

Abbreviations

“BHQ,” as used herein, refers generally to dark quenchers including oneor more diazo bond and specifically to “Black Hole Quenchers™.”Exemplary BHQ's are described in U.S. Pat. No. 7,019,129. “FET,” as usedherein, refers to “Fluorescence Energy Transfer.” “FRET,” as usedherein, refers to “Fluorescence Resonance Energy Transfer.” These termsare used herein to refer to both radiative and non-radiative energytransfer processes. For example, processes in which a photon is emittedand those involving long range electron transfer are included withinthese terms. Throughout this specification, both of these phenomena aresubsumed under the general term “donor-acceptor energy transfer.” “SNP”refers to “Single Nucleotide Polymorphism.”

Definitions

Before the invention is described in greater detail, it is to beunderstood that the invention is not limited to particular embodimentsdescribed herein as such embodiments may vary. It is also to beunderstood that the terminology used herein is for the purpose ofdescribing particular embodiments only, and the terminology is notintended to be limiting. The scope of the invention will be limited onlyby the appended claims. Unless defined otherwise, all technical andscientific terms used herein have the same meaning as commonlyunderstood by one of ordinary skill in the art to which this inventionbelongs. Where a range of values is provided, it is understood that eachintervening value, to the tenth of the unit of the lower limit unlessthe context clearly dictates otherwise, between the upper and lowerlimit of that range and any other stated or intervening value in thatstated range, is encompassed within the invention. The upper and lowerlimits of these smaller ranges may independently be included in thesmaller ranges and are also encompassed within the invention, subject toany specifically excluded limit in the stated range. Where the statedrange includes one or both of the limits, ranges excluding either orboth of those included limits are also included in the invention.Certain ranges are presented herein with numerical values being precededby the term “about.” The term “about” is used herein to provide literalsupport for the exact number that it precedes, as well as a number thatis near to or approximately the number that the term precedes. Indetermining whether a number is near to or approximately a specificallyrecited number, the near or approximating unrecited number may be anumber, which, in the context in which it is presented, provides thesubstantial equivalent of the specifically recited number. Allpublications, patents, and patent applications cited in thisspecification are incorporated herein by reference to the same extent asif each individual publication, patent, or patent application werespecifically and individually indicated to be incorporated by reference.Furthermore, each cited publication, patent, or patent application isincorporated herein by reference to disclose and describe the subjectmatter in connection with which the publications are cited. The citationof any publication is for its disclosure prior to the filing date andshould not be construed as an admission that the invention describedherein is not entitled to antedate such publication by virtue of priorinvention. Further, the dates of publication provided might be differentfrom the actual publication dates, which may need to be independentlyconfirmed.

It is noted that the claims may be drafted to exclude any optionalelement. As such, this statement is intended to serve as antecedentbasis for use of such exclusive terminology as “solely,” “only,” and thelike in connection with the recitation of claim elements, or use of a“negative” limitation. As will be apparent to those of skill in the artupon reading this disclosure, each of the individual embodimentsdescribed and illustrated herein has discrete components and featureswhich may be readily separated from or combined with the features of anyof the other several embodiments without departing from the scope orspirit of the invention. Any recited method may be carried out in theorder of events recited or in any other order that is logicallypossible. Although any methods and materials similar or equivalent tothose described herein may also be used in the practice or testing ofthe invention, representative illustrative methods and materials are nowdescribed.

The following definitions are broadly applicable to each of theembodiments of the present invention set forth herein below. Unlessdefined otherwise, all technical and scientific terms used hereingenerally have the same meaning as commonly understood by one ofordinary skill in the art to which this invention belongs. Generally,the nomenclature used herein and the laboratory procedures in cellculture, molecular genetics, organic chemistry and nucleic acidchemistry and hybridization described below are those well-known andcommonly employed in the art. Standard techniques are used for nucleicacid and peptide synthesis. Molecular biological techniques andprocedures are generally performed according to conventional methods inthe art and various general references (see generally, Sambrook et al.MOLECULAR CLONING: A LABORATORY MANUAL, 2d ed. (1989) Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y., which is incorporated hereinby reference). The nomenclature used herein and the laboratoryprocedures in analytical chemistry, and organic synthesis are thosewell-known and commonly employed in the art. Standard techniques, ormodifications thereof, are used for chemical syntheses and chemicalanalyses.

The term “alkyl,” by itself or as part of another substituent, means,unless otherwise stated, a straight- or branched-chain, or cyclichydrocarbon radical, or combination thereof, which may be fullysaturated, mono- or polyunsaturated and can include mono-, di-, tri- andtetra-valent radicals, having the number of carbon atoms designated(i.e. C₁-C₁₀ means one to ten carbons). Examples of saturatedhydrocarbon radicals include, but are not limited to, groups such asmethyl, ethyl, n-propyl, isopropyl, n-butyl, t-butyl, isobutyl,sec-butyl, cyclohexyl, (cyclohexyl)methyl, cyclopropylmethyl, homologsand isomers of, for example, n-pentyl, n-hexyl, n-heptyl, n-octyl, andthe like. An unsaturated alkyl group is one having one or more doublebonds or triple bonds. Examples of unsaturated alkyl groups include, butare not limited to, vinyl, 2-propenyl, crotyl, 2-isopentenyl,2-(butadienyl), 2,4-pentadienyl, 3-(1,4-pentadienyl), ethynyl, 1- and3-propynyl, 3-butynyl, and the higher homologs and isomers. The term“alkyl,” unless otherwise noted, also optionally include thosederivatives of alkyl defined in more detail below, such as“heteroalkyl.” Alkyl groups that are limited to hydrocarbon groups aretermed “homoalkyl”. The term “alkyl”, as used herein refers to alkyl,alkenyl and alkynyl moieties, each of which can be mono-, di- orpolyvalent species as appropriate to satisfy valence requirements. Alkylgroups are optionally substituted, e.g., with one or more groupsreferred to herein as an “alkyl group substituent.”

The term “alkylene” by itself or as part of another substituent means adivalent radical derived from an alkyl moiety, as exemplified, but notlimited, by —CH₂CH₂CH₂CH₂—, and further includes those groups describedbelow as “heteroalkylene.” Typically, an alkyl (or alkylene) group willhave from 1 to 24 carbon atoms, with those groups having 10 or fewercarbon atoms being preferred in the present invention. For alkylene andheteroalkylene linker groups, it is optional that no orientation of thelinker group is implied by the direction in which the formula of thelinker group is written. For example, the formula —C(O)₂R′— represents—C(O)₂R′— and, optionally, —R′C(O)₂—. A “lower alkyl” or “loweralkylene” is a shorter chain alkyl or alkylene group, generally havingeight, seven, six, five or fewer carbon atoms.

The terms “alkoxy,” “alkylamino” and “alkylthio” (or thioalkoxy) areused in their conventional sense, and refer to those alkyl groupsattached to the remainder of the molecule via an oxygen atom, an aminogroup, or a sulfur atom, respectively.

The term “heteroalkyl,” by itself or in combination with another term,means, unless otherwise stated, a stable straight- or branched-chain, orcyclic alkyl radical consisting of the stated number of carbon atoms andat least one heteroatom selected from the group consisting of B, O, N,Si and S, wherein the heteroatom may optionally be oxidized and thenitrogen atom may optionally be quaternized. The heteroatom(s) may beplaced at any internal position of the heteroalkyl group or at aterminus of the chain, e.g., the position through which the alkyl groupis attached to the remainder of the molecule. Examples of “heteroalkyl”groups include, but are not limited to, —CH₂—CH₂—O—CH₃, —CH₂—CH₂—NH—CH₃,—CH₂—CH₂—N(CH₃)—CH₃, —CH₂—S—CH₂—CH₃, —CH₂—CH₂, —S(O)—CH₃,—CH₂—CH₂—S(O)₂—CH₃, —CH═CH—O—CH₃, —Si(CH₃)₃, —CH₂—CH═N—OCH₃, and—CH═CH—N(CH₃)—CH₃. Two or more heteroatoms may be consecutive, such as,for example, —CH₂—NH—OCH₃ and —CH₂—O—Si(CH₃)₃. Similarly, the term“heteroalkylene” by itself or as part of another substituent refers to asubstituted or unsubstituted divalent heteroalkyl radical, asexemplified, but not limited by, —CH₂—CH₂—S—CH₂—CH₂— and—CH₂—S—CH₂—CH₂—NH—CH₂—. For heteroalkylene groups, heteroatoms can alsooccupy either or both of the chain termini (e.g., alkyleneoxy,alkylenedioxy, alkyleneamino, alkylenediamino, and the like).

The terms “cycloalkyl” and “heterocycloalkyl”, by themselves or incombination with other terms, represent, unless otherwise stated, cyclicversions of “alkyl” and “heteroalkyl”, respectively. Additionally, forheterocycloalkyl, a heteroatom can occupy the position at which theheterocycle is attached to the remainder of the molecule. Examples ofcycloalkyl include, but are not limited to, cyclopentyl, cyclohexyl,1-cyclohexenyl, 3-cyclohexenyl, cycloheptyl, and the like. Examples ofheterocycloalkyl include, but are not limited to,1-(1,2,5,6-tetrahydropyridyl), 1-piperidinyl, 2-piperidinyl,3-piperidinyl, 4-morpholinyl, 3-morpholinyl, tetrahydrofuran-2-yl,tetrahydrofuran-3-yl, tetrahydrothien-2-yl, tetrahydrothien-3-yl,1-piperazinyl, 2-piperazinyl, and the like.

The terms “halo” or “halogen,” by themselves or as part of anothersubstituent, mean, unless otherwise stated, a fluorine, chlorine,bromine, or iodine atom. Additionally, terms such as “haloalkyl,” aremeant to include monohaloalkyl and polyhaloalkyl. For example, the term“halo(C₁-C₄)alkyl” is meant to include, but not be limited to,trifluoromethyl, 2,2,2-trifluoroethyl, 4-chlorobutyl, 3-bromopropyl, andthe like.

The term “aryl” means, unless otherwise stated, a polyunsaturated,aromatic, substituent that can be a single ring or multiple rings(preferably from 1 to 3 rings, one or more of which is optionally acycloalkyl or heterocycloalkyl), which are fused together or linkedcovalently. The term “heteroaryl” refers to aryl groups (or rings) thatcontain from one to four heteroatoms selected from N, O, and S, whereinthe nitrogen and sulfur atoms are optionally oxidized, and the nitrogenatom(s) are optionally quaternized. A heteroaryl group can be attachedto the remainder of the molecule through a heteroatom. Non-limitingexamples of aryl and heteroaryl groups include phenyl, 1-naphthyl,2-naphthyl, 4-biphenyl, 1-pyrrolyl, 2-pyrrolyl, 3-pyrrolyl, 3-pyrazolyl,2-imidazolyl, 4-imidazolyl, pyrazinyl, 2-oxazolyl, 4-oxazolyl,2-phenyl-4-oxazolyl, 5-oxazolyl, 3-isoxazolyl, 4-isoxazolyl,5-isoxazolyl, 2-thiazolyl, 4-thiazolyl, 5-thiazolyl, 2-furyl, 3-furyl,2-thienyl, 3-thienyl, 2-pyridyl, 3-pyridyl, 4-pyridyl, 2-pyrimidyl,4-pyrimidyl, 5-benzothiazolyl, purinyl, 2-benzimidazolyl, 5-indolyl,1-isoquinolyl, 5-isoquinolyl, 2-quinoxalinyl, 5-quinoxalinyl,3-quinolyl, and 6-quinolyl. Substituents for each of the above notedaryl and heteroaryl ring systems are selected from the group of “arylgroup substituents” described below.

For brevity, the term “aryl” when used in combination with other terms(e.g., aryloxy, arylthioxy, arylalkyl) optionally includes both homoaryland heteroaryl rings as defined above. Thus, the term “arylalkyl”optionally includes those radicals in which an aryl group is attached toan alkyl group (e.g., benzyl, phenethyl, pyridylmethyl and the like)including those alkyl groups in which a carbon atom (e.g., a methylenegroup) has been replaced by, for example, an oxygen atom (e.g.,phenoxymethyl, 2-pyridyloxymethyl, 3-(1-naphthyloxy)propyl, and thelike).

Substituents for the alkyl and heteroalkyl radicals (including thosegroups often referred to as alkylene, alkenyl, heteroalkylene,heteroalkenyl, alkynyl, cycloalkyl, heterocycloalkyl, cycloalkenyl, andheterocycloalkenyl) are generically referred to as “alkyl groupsubstituents,” and they can be one or more of a variety of groupsselected from, but not limited to: —OR′, ═O, ═NR′, ═N—OR′, —NR′R″, —SR′,-halogen, —SiR′R″R′″, —OC(O)R′, —C(O)R′, —CO₂R′, —CONR′R″, —OC(O)NR′R″,—NR″C(O)R′, —NR′—C(O)NR″R′″, —NR″C(O)₂R′, —NR—C(NR′R″R′″)═NR″″,—NR—C(NR′R″)═NR′″, —S(O)R′, —S(O)₂R′, —S(O)₂NR′R″, —NRSO₂R′, —CN and—NO₂ in a number ranging from zero to (2m′+1), where m′ is the totalnumber of carbon atoms in such radical. R′, R″, R′″ and R″″ eachpreferably independently refer to hydrogen, substituted or unsubstitutedheteroalkyl, substituted or unsubstituted aryl, e.g., aryl substitutedwith 1-3 halogens, substituted or unsubstituted alkyl, alkoxy orthioalkoxy groups, or arylalkyl groups. When a compound of the inventionincludes more than one R group, for example, each of the R groups isindependently selected as are each R′, R″, R′″ and R″″ groups when morethan one of these groups is present. When R′ and R″ are attached to thesame nitrogen atom, they can be combined with the nitrogen atom to forma 5-, 6-, or 7-membered ring. For example, —NR′R″ is meant to include,but not be limited to, 1-pyrrolidinyl and 4-morpholinyl. From the abovediscussion of substituents, one of skill in the art will understand thatthe term “alkyl” includes groups with carbon atoms bound to groups otherthan hydrogen, such as haloalkyl (e.g., —CF₃ and —CH₂CF₃) and acyl(e.g., —C(O)CH₃, —C(O)CF₃, —C(O)CH₂OCH₃, and the like). Exemplary alkylgroup substituents include those groups referred to herein as “reactivefunctional groups” and “linkage sites.” In various embodiments, thealkyl group substituent is a phosphorus-containing moiety, e.g., aphosphodiester or a phosphodiester modification such as those describedherein.

Similar to the substituents described for the alkyl radical,substituents for the aryl and heteroaryl groups are generically referredto as “aryl group substituents.” Exemplary substituents are selectedfrom the list of alkyl group substituents and others, for example:halogen, —OR′, ═O, ═NR′, ═N—OR′, —NR′R″, —SR′, —SiR′R″R′″, —OC(O)R′,—C(O)R′, —CO₂R′, —CONR′R″, —OC(O)NR′R″, —NR″C(O)R′, —NR′—C(O)NR″R′″,—NR″ C(O)₂R′, —NR—C(NR′R″R′″)═NR″″, —NR—C(NR′R″)═NR′″, —S(O)R′,—S(O)₂R′, —S(O)₂NR′R″, —NRSO₂R′, —CN and —NO₂, —R′, —N₃, —CH(Ph)₂,fluoro(C₁-C₄)alkoxy, and fluoro(C₁-C₄)alkyl, in a number ranging fromzero to the total number of open valences on the aromatic ring system;and where R′, R″, R′″ and R″″ are preferably independently selected fromhydrogen, substituted or unsubstituted alkyl, substituted orunsubstituted heteroalkyl, substituted or unsubstituted aryl andsubstituted or unsubstituted heteroaryl. When a compound of theinvention includes more than one R group, for example, each of the Rgroups is independently selected as are each R′, R″, R′″ and R″″ groupswhen more than one of these groups is present.

Two of the substituents on adjacent atoms of the aryl or heteroaryl ringmay optionally be replaced with a substituent of the formula-T-C(O)—(CRR′)_(q)—U—, wherein T and U are independently —NR—, —O—,—CRR′— or a single bond, and q is an integer from 0 to 3. Alternatively,two of the substituents on adjacent atoms of the aryl or heteroaryl ringmay optionally be replaced with a substituent of the formula-A-(CH₂)_(r)—B—, wherein A and B are independently —CRR′—, —O—, —NR—,—S—, —S(O)—, —S(O)₂—, —S(O)₂NR′— or a single bond, and r is an integerof from 1 to 4. One of the single bonds of the new ring so formed mayoptionally be replaced with a double bond. Alternatively, two of thesubstituents on adjacent atoms of the aryl or heteroaryl ring mayoptionally be replaced with a substituent of the formula—(CRR′)_(s)—X—(CR″R′″)_(d)—, where s and d are independently integers offrom 0 to 3, and X is —O—, —NR′—, —S—, —S(O)—, —S(O)₂—, or —S(O)₂NR′—.The substituents R, R′, R″ and R″ are preferably independently selectedfrom hydrogen or substituted or unsubstituted (C₁-C₁₆)alkyl. Exemplaryaryl group substituents include those groups referred to herein as“reactive functional groups” and “linkage sites.”

As used herein, the term “heteroatom” includes oxygen (O), nitrogen (N),sulfur (S) and silicon (Si).

The symbol “R” is a general abbreviation that represents a substituentgroup that is selected from H, substituted or unsubstituted alkyl,substituted or unsubstituted heteroalkyl, substituted or unsubstitutedaryl, substituted or unsubstituted heteroaryl, and substituted orunsubstituted heterocyclyl groups. R can also refer to alkyl groupsubstituents and aryl group substituents.

The term “salt(s)” includes salts of the compounds which are preparedwith relatively nontoxic acids or bases, depending on the particularsubstituents found on the compounds described herein. When compounds ofthe present invention contain relatively acidic functionalities, baseaddition salts can be obtained by contacting the neutral form of suchcompounds with a sufficient amount of the desired base, either neat orin a suitable inert solvent. Examples of base addition salts includesodium, potassium, calcium, ammonium, organic amino, or magnesium salt,or a similar salt. When compounds of the present invention containrelatively basic functionalities, acid addition salts can be obtained bycontacting the neutral form of such compounds with a sufficient amountof the desired acid, either neat or in a suitable inert solvent.Examples of acid addition salts include those derived from inorganicacids like hydrochloric, hydrobromic, nitric, carbonic,monohydrogencarbonic, phosphoric, monohydrogenphosphoric,dihydrogenphosphoric, sulfuric, monohydrogensulfuric, hydriodic, orphosphorous acids, and the like, as well as the salts derived fromrelatively nontoxic organic acids like acetic, propionic, isobutyric,butyric, maleic, malic, malonic, benzoic, succinic, suberic, fumaric,lactic, mandelic, phthalic, benzenesulfonic, p-tolylsulfonic, citric,tartaric, methanesulfonic, and the like. Also included are salts ofamino acids such as arginate, and the like, and salts of organic acidslike glucuronic or galactunoric acids and the like (see, for example,Berge et al., Journal of Pharmaceutical Science, 66: 1-19 (1977)).Certain specific compounds of the present invention contain both basicand acidic functionalities that allow the compounds to be converted intoeither base or acid addition salts. Hydrates of the salts are alsoincluded.

As used herein, “nucleic acid” means nucleosides, nucleotides andoligonucleotides, e.g., DNA, RNA, whether single-stranded,double-stranded, or in more highly aggregated hybridization motifs, andany chemical modifications thereof. Modifications include, but are notlimited to, those providing chemical groups that incorporate additionalcharge, polarizability, hydrogen bonding, electrostatic interaction, andreactivity to the nucleic acid ligand nucleobases or to the nucleic acidligand as a whole. Such modifications include, but are not limited to,phosphodiester group modifications (e.g., phosphorothioates,methylphosphonates), sugar modifications, 5-position pyrimidinemodifications, 8-position purine modifications, modifications atexocyclic amines, substitution with non-standard or non-naturalnucleobases such as 4-thiouridine, 5-bromo or 5-iodo-uracil; backbonemodifications such as peptide nucleic acids (PNAs), glycol nucleic acids(GNAs), morpholinos; methylations such as 2′-O-methyl nucleosides,5-methyl-2′-deoxycytidine; unusual base-pairing combinations such as theisobases, isocytidine and isoguanidine and the like. A “nucleomonomer”refers to a single nucleic acid unit, which can be a nucleoside,nucleotide or a modification thereof.

“Nucleobase” as used herein includes those moieties which contain notonly the known purine and pyrimidine heterocycles, but also heterocycleanalogs and tautomers thereof. Purines include adenine, guanine andxanthine and exemplary purine analogs include 8-oxo-N⁶-methyladenine and7-deazaxanthine. Pyrimidines include uracil and cytosine and theiranalogs such as 5-methylcytosine, 5-methyluracil and 4,4-ethanocytosine.This term also encompasses non-natural nucleobases. Representativenon-natural nucleobases include 5-fluorouracil, 5-bromouracil,5-chlorouracil, 5-iodouracil, hypoxanthine, xanthine, 4-acetylcytosine,5-(carboxyhydroxylmethyl) uracil,5-carboxymethylaminomethyl-2-thiouridine,5-carboxymethylaminomethyluracil, dihydrouracil,beta-D-galactosylqueosine, inosine, N⁶-isopentenyladenine,1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine,2-methylguanine, 3-methylcytosine, 5-methylcytosine, N⁶-adenine,7-methylguanine, 5-methylaminomethyluracil,5-methoxyaminomethyl-2-thiouracil, beta-D-mannosylqueosine,5′-methoxycarboxymethyluracil, 5-methoxyuracil,2-methylthio-N⁶-isopentenyladenine, uracil-5-oxyacetic acid (v),wybutoxosine, pseudouracil, queosine, 2-thiocytosine,5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil,uracil-5-oxyacetic acid methyl ester, uracil-5-oxyacetic acid (v),5-methyl-2-thiouracil, 3-(3-amino-3-N-2-carboxypropyl) uracil, (acp3)w,nitroindole, and 2,6-diaminopurine.

In various embodiments, the inventive compounds include pyrimidinesderivatized at the 5-position. The derivatives are 1-alkenyl-,1-alkynyl-, heteroaromatic- and 1-alkynyl-heteroaromatic modifications.“1-Alkenyl” means an olefinically-unsaturated (double bond containing)acyclic group. “1-Alkynyl” means an acetylenically-unsaturated (triplebond containing) acylic group

As used herein, “nucleoside” means a subset of nucleic acid in which anucleobase is covalently attached to a sugar or sugar analog and whichoptionally includes a phosphite, phosphoramidite or phosphine. The termnucleoside includes ribonucleosides, deoxyribonucleosides, or any othernucleoside which is an N-glycoside or C-glycoside of a nucleobase. Thestereochemistry of the sugar carbons can be other than that of D-ribose.Nucleosides also include those species which contain modifications ofthe sugar moiety, for example, wherein one or more of the hydroxylgroups are replaced with a halogen, a heteroatom, an aliphatic groups,or are functionalized as ethers, amines, thiols, and the like. Thepentose moiety can be replaced by a hexose or an alternate structuresuch as a cyclopentane ring, a 6-member morpholino ring and the like.Nucleosides as defined herein also include a nucleobase linked to anamino acid and/or an amino acid analog having a free carboxyl groupand/or a free amino group and/or protected forms thereof. Nucleosidesalso optionally include one or more nucleobase modification, e.g.,modified with a fluorocarbyl, alkenyl or alkynyl moiety. A nucleosideincluding a phosphodiester or phosphodiester modification, is referredto herein as a nucleotide.

“Sugar modification,” as used herein, means any pentose or hexose moietyother than 2′-deoxyribose. Modified sugars include, for example,D-ribose, 2′-O-alkyl, 2′-amino, 2′-halo functionalized pentoses, hexosesand the like. Exemplary sugar modifications include those sugars inwhich one or more of the hydroxyl groups is replaced with a halogen, aheteroatom, an alkyl moiety, or are functionalized as ethers, esters,and the like. The pentose moiety can be replaced by a hexose or analternate structure such as a cyclopentane ring, a 6-member morpholinoring and the like. Nucleosides as defined herein are also intended toinclude a nucleobase linked to an amino acid and/or an amino acid analoghaving a free carboxyl group and/or a free amino group and/or protectedforms thereof. Sugars having a stereochemistry other than that of aD-ribose are also included.

“Phosphodiester group modification” means any analog of the nativephosphodiester group that covalently couples adjacent nucleomonomers.Substitute linkages include phosphodiester analogs, e.g. such asphosphorothioate and methylphosphonate, and nonphosphorus containinglinkages, e.g. such as acetals and amides.

Nucleic acid modification also include 3′, 5′, and base modificationssuch as labeling with a quencher (e.g., a BHQ), a fluorophore,intercalator, minor groove binder, a fluorocarbon, a stabilizing groupor another moiety. In various embodiments, the modification or label iscovalently conjugated to the oligomer through a linker group.

Oligomers are defined herein as two or more nucleomonomers covalentlycoupled to each other by a phosphodiesester or modified phosphodiestermoiety. Thus, an oligomer can have as few as two nucleomonomers (adimer), and have essentially no upper limit of nucleomonomers. Oligomerscan be binding competent and, thus, can base pair with cognatesingle-stranded or double-stranded (or higher order aggregation) nucleicacid sequences. Oligomers are also useful as synthons for longeroligomers as described herein. Oligomers can also contain abasic sitesand pseudonucleosides. In various embodiments, the oligomers of theinvention are functionalized. The moieties functionalizing the oligomersare discussed below. In describing certain embodiments the term“oligomer” is used interchangeably to refer to the nucleic acid sequenceof the oligomer, the modified nucleic acid sequence providing a probe ofthe invention or the modified nucleic acid sequence providing a solidsupport of the invention.

“Peptide” refers to an oligomer in which the monomers are amino acidsand are joined together through amide bonds, alternatively referred toas a polypeptide. When the amino acids are α-amino acids, either theL-optical isomer or the D-optical isomer can be used. Additionally,unnatural amino acids, for example, β-alanine, phenylglycine andhomoarginine are also included. Commonly encountered amino acids thatare not gene-encoded may also be used in the present invention. All ofthe amino acids used in the present invention may be either the D- orL-isomer. The L-isomers are generally preferred. In addition, otherpeptidomimetics are also useful in the present invention. For a generalreview, see, Spatola, A. F., in CHEMISTRY AND BIOCHEMISTRY OF AMINOACIDS, PEPTIDES AND PROTEINS, B. Weinstein, eds., Marcel Dekker, NewYork, p. 267 (1983).

A “solid support” is a solid material having a surface for attachment ofmolecules, compounds, cells, or other entities, or to which surface suchspecies are attached. The surface of a solid support can be flat orotherwise configured. A solid support can be porous or non-porous. Asolid support can be a chip or array that comprises a surface, and thatmay comprise glass, silicon, nylon, polymers, plastics, ceramics, ormetals. A solid support can also be a membrane, such as a nylon,nitrocellulose, or polymeric membrane, or a plate or dish and can becomprised of glass, ceramics, metals, or plastics, such as, for example,a 96-well plate made of, for example, polystyrene, polypropylene,polycarbonate, or polyallomer. A solid support can also be a bead orparticle of any shape, and is preferably spherical or nearly spherical,and preferably a bead or particle has a diameter or maximum width of 1millimeter or less, more preferably of between 0.1 to 100 microns. Suchparticles or beads can be comprised of any suitable material, e.g.,glass or ceramics, and/or one or more polymers, such as, for example,nylon, polytetrafluoroethylene, TEFLON™, polystyrene, polyacrylamide,sepharose, agarose, cellulose, cellulose derivatives, or dextran, and/orcan comprise metals, particularly paramagnetic metals, such as iron.

Supports for solid phase synthesis are known in the art and include, butare not limited to, high cross-linker polystyrene (McCollum, et al.,Tetrahedron Lett. 32: 4069-4072 (1991), polystyrene/PEG copolymer (Gao,et al., Tetrahedron Lett. 32: 5477-5480 (1991), silica gel (Chow, etal., Nucl. Acids Res. 9: 2807-2817 (1981)), polyamide bonded silica gel(Gait, et al., Nucl. Acids Res. 10: 6243-6254 (1982)), cellulose (Crea,et al., Nucl. Acids Res. 8: 2331-2348 (1980)), and controlled pore glass(CPG) (Koster, et al., Tetrahedron Lett. 24: 747-750 (1983). Anexemplary solid support is CPG beads. CPG beads can be derivatized forthe attachment of a nucleomonor or oligomer in a variety of ways. Forexample, CPG beads can be treated with 3-aminopropyltriethoxysilane toadd an amino propyl linker handle for the attachment of oligonucleotideanalogue monomers or dimers (Koster, et al., Tetrahedron Lett. 24:747-750 (1983), or, preferably, a long-chain alkylamine group, mostpreferably including a terminal nucleoside, can be attached to CPG(Adams, et al., J. Am. Chem. Soc. 105: 661-663 (1983)). Supports foroligonucleotide synthesis or peptide synthesis, for example dT-LCAA-CPG(Applied Biosystems), are commercially available.

An “intercalator” refers to a planar aromatic or heteroaromatic moietythat is capable of partial insertion and stacking between adjacentnucleobases. These moieties may be small molecules or part of a largerentity, such as a protein. Non-limiting examples of intercalatorsinclude acridines, anthracenes, anthracyclines, anthracyclinone,methylene blue, indole, anthraquinone, quinoline, isoquinoline,dihydroquinones, tetracyclines, psoralens, coumarins, ethidium halides,ethidium homodimers, homodimeric oxazole yellow (YOYO), thiazole orange(TOTO), dynemicins, 1,10-phenanthroline-copper, calcheamicin,porphyrins, distamycins, netropcins, and viologens.

A “minor groove binder” refers to a moiety typically having a molecularweight of approximately 150 to approximately 2000 Daltons. The moietybinds in a non-intercalating manner into the minor groove of doublestranded (or higher order aggregation) DNA, RNA or hybrids thereof,preferably, with an association constant greater than approximately 10³M⁻¹. Minor groove binding compounds have widely varying chemicalstructures, however, exemplary minor groove binders have a crescentshape three dimensional structure. Exemplar include certain naturallyoccurring compounds such as netropsin, distamycin and lexitropsin,mithramycin, chromomycin A₃, olivomycin, anthramycin, sibiromycin, aswell as further related antibiotics and synthetic derivatives. Certainbisquarternary ammonium heterocyclic compounds, diarylamidines such aspentamidine, stilbamidine and berenil, CC-1065 and related pyrroloindoleand indole polypeptides, Hoechst 33258, 4′-6-diamidino-2-phenylindole(DAPI) as well as a number of oligopeptides consisting of naturallyoccurring or synthetic amino acids are minor groove binder compounds.Exemplary minor groove binders are described in U.S. Pat. No. 6,084,102.This type of binding can be detected by well establishedspectrophotometric methods, such as ultraviolet (u.v.) and nuclearmagnetic resonance (NMR) spectroscopy and also by gel electrophoresis.Shifts in u.v. spectra upon binding of a minor groove binder molecule,and NMR spectroscopy utilizing the “Nuclear Overhauser” (NOSEY) effectare particularly well known and useful techniques for this purpose. Gelelectrophoresis detects binding of a minor groove binder to doublestranded DNA or fragment thereof, because upon such binding the mobilityof the double stranded DNA changes.

The minor groove binder is typically attached to the oligomer or solidsupport through a linker comprising a chain about 20, about 15 atoms,about 10 or about 5 atoms.

Intercalating moieties or agents are readily distinguished from minorgroove binders on the basis that the intercalating agents are flataromatic (preferably polycyclic) molecules versus the “crescent shape”or analogous geometry of the minor groove binders. An experimentaldistinction can also be made by NMR spectroscopy utilizing the NuclearOverhauser effect.

The term “linker” or “L”, as used herein, refers to a single covalentbond (“zero-order”) or a series of stable covalent bonds incorporating1-30 nonhydrogen atoms selected from the group consisting of C, N, O, S,Si and P that covalently link together the components of the compoundsof the invention, e.g., linking a solid support to a stabilizing agent,a quencher, a nucleomonomer or oligomer of the invention; or linking aquencher or stabilizing moiety to a nucleobase in an amidite of theinvention. Exemplary linkers include 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29or 30 non-hydrogen atoms. Unless otherwise specified, “linking,”“linked,” “linkage,” “conjugating,” “conjugated” and analogous termsrelating to attachment refer to techniques utilizing and speciesincorporating linkers. Exemplary linkers include a linkage site asdefined herein. Moreover, a linker is of use to attach an oligomer ornascent oligomer (during oligomer synthesis) to the solid support of theinvention. Thus, the invention also provides an oligomer of theinvention covalently attached to a solid support (e.g., a solid supportof the invention) through a linker. The solid supports and oligomers ofthe invention optionally include a cleavable linker between twocomponents of the solid support and oligomer (e.g., between the oligomerand the solid support, between the fluorophore and oligomer, between thequencher and oligomer, between the fluorophore and quencher, etc.). Invarious embodiments, the linker joining the solid support to theoligomer is a cleavable linker.

A “cleavable linker” is a linker that has one or more cleavable groupsthat may be broken by the result of a reaction or condition. Anexemplary cleavable linker is located within R⁸ of Formula I or II,serving to allow for the expedient separation of a synthesized oligomerof the invention from the solid support upon which it was synthesized.The term “cleavable group” refers to a moiety that allows for release ofa component of the solid support or oligomer of the invention bycleaving a bond linking the released moiety to the remainder of theconjugate. Exemplary cleavage mechanisms of use both in preparing andusing the oligomers and solid supports of the invention areenzymatically or otherwise chemically mediated.

In addition to enzymatically cleavable groups, it is within the scope ofthe present invention to include one or more sites that are cleaved bythe action of an agent other than an enzyme. Exemplary non-enzymaticcleavage agents include, but are not limited to, acids, bases, light(e.g., nitrobenzyl derivatives, phenacyl groups, ortho-hydroxcinnamateesters, benzoin esters), and heat. Many cleaveable groups are known inthe art. See, for example, Jung et al., Biochem. Biophys. Acta, 761:152-162 (1983); Joshi et al., J. Biol. Chem., 265: 14518-14525 (1990);Zarling et al., J. Immunol., 124: 913-920 (1980); Bouizar et al., Eur.J. Biochem., 155: 141-147 (1986); Park et al., J. Biol. Chem., 261:205-210 (1986); Browning et al., J. Immunol., 143: 1859-1867 (1989).Moreover a broad range of cleavable, bifunctional (both homo- andhetero-bifunctional) spacer arms are commercially available.

An exemplary cleavable group is cleavable by a reagent, e.g. sodiumhydroxide, ammonia or other amine. In various embodiments the cleavablelinker is readily cleaved at room temperature or under heat. In oneembodiment, R⁸ of Formula I or II comprises a cleavable linker that iscleaved by treatment with an amine, e.g., ammonia or an essentiallyanhydrous amine in an organic solvent.

A “linkage site,” is a moiety that connects two or more components(e.g., functional component, solid support, oligonucleotide, or linker).This term refers to a covalent bond that is formed by reaction ofcomplementary reaction partners, each of which has a functional group ofcomplementary reactivity to that of its partner. Linkage sites in thesolid support and oligomers of the invention are independently selected.Exemplary linkage sites include, but are not limited to S, SC(O)NH,HNC(O)S, SC(O)O, O, NH, NHC(O), (O)CNH and NHC(O)O, and OC(O)NH, CH₂S,CH₂O, CH₂CH₂O, CH₂CH₂S, (CH₂)_(o)O, (CH₂)_(o)S or (CH₂)_(o)Y^(x)-PEGwherein, Y^(x) is S, NH, NHC(O), C(O)NH, NHC(O)O, OC(O)NH, or O and o isan integer from 1 to 50. In each of these exemplary linkage sites, NHcan be NR^(t) in which R^(t) is substituted or unsubstituted alkyl orsubstituted or unsubstituted heteroalkyl. A linkage site can also be aphosphodiester group. In various embodiments, the linkage site isbetween a linker and a fluorophore, a linker and a quencher, a linkerand a stabilizing moiety or a linker and a solid support. In anexemplary embodiment of the oligomers and solid support of theinvention, each linkage site is different.

The term “fluorophore” as used herein refers to a moiety that isinherently fluorescent or demonstrates a change in fluorescence uponbinding to a biological compound or metal ion, or metabolism by anenzyme, i.e., fluorogenic. Fluorophores may be substituted to alter thesolubility, spectral properties or physical properties of thefluorophore. Numerous fluorophores are known to those skilled in the artand include, but are not limited to coumarins, acridines, furans,dansyls, cyanines, pyrenes, naphthalenes, benzofurans, quinolines,quinazolinones, indoles, benzazoles, borapolyazaindacenes, oxazines andxanthenes, with the latter including fluoresceins, rhodamines, rosaminesand rhodols. These and other fluorophores of use in the invention aredescribed in Haugland, MOLECULAR PROBES HANDBOOK OF FLUORESCENT PROBESAND RESEARCH CHEMICALS. Further useful fluorophores are described incommonly owned U.S. Patent Application Publication No. 2005/0214833 and2005/0170363 and herein below.

As used herein, “quencher” refers to any fluorescence-modifying moietyof the invention that can attenuate at least partly the light emitted bya fluorophore. This attenuation is referred to herein as “quenching”.Hence, in various embodiments, excitation of the fluorophore in thepresence of the quenching group leads to an emission signal that is lessintense than expected, or even completely absent. Quenching typicallyoccurs through energy transfer between the excited fluorophore and thequenching group.

The fluorophore or quencher may include substituents enhancing adesirable property, e.g., solubility in water, cell permeability or analtered absorption and emission spectrum, relative to the “parent”compound in the absence of such substituent. As such the fluorophore orquencher of use in the invention include substituents that enhance adesirable property relative to an identical parent compound in theabsence of the improving substituent.

A “functional component” is a generic term for a moiety in a compound ofthe invention having a structure selected from a quencher, a fluorophoreor a stabilizing moiety (including, but not limited to, intercalators,minor groove binding moieties, nucleobases modified with a stabilizingmoiety (e.g., alkynyl moieties, and fluoroalkyl moieties), andconformational stabilizing moieties, such as those described in commonlyowned U.S. Patent Application Publication No. 2007/0059752).

The expression “amplification of polynucleotides” includes but is notlimited to methods such as polymerase chain reaction (PCR), ligationamplification (or ligase chain reaction, LCR) and amplification methodsbased on the use of Q-beta replicase. These methods are well known andwidely practiced in the art. See, e.g., U.S. Pat. Nos. 4,683,195 and4,683,202 and Innis et al., 1990 (for PCR); and Wu et al., 1989a (forLCR). Reagents and hardware for conducting PCR are commerciallyavailable. Primers useful to amplify sequences from a particular generegion are preferably complementary to, and hybridize specifically tosequences in the target region or in its flanking regions. Nucleic acidsequences generated by amplification may be sequenced directly.Alternatively the amplified sequence(s) may be cloned prior to sequenceanalysis. A method for the direct cloning and sequence analysis ofenzymatically amplified genomic segments has been described by Scharf(1986). The present invention provides oligomeric primers of use inamplification processes. Moreover, there is provided a solid support ofuse in synthesizing such primers. In addition to primers, the inventionprovides probes, and methods of using such probes, to detect,characterize and/or quantify the products of amplification: alsoprovided are solid supports of use to synthesize these oligomericprobes.

The term “base-stacking perturbations” refers to any event that causes aperturbation in base-stacking such as, for example, a base-pairmismatch, a protein binding to its recognition site, or any otherentities that form oligonucleotide adducts. Various probes of theinvention are capable of detecting, characterizing and/or quantifyingsuch base-stacking perturbations. Moreover, the invention provides solidsupports of use in synthesizing probes capable of detecting,characterizing and/or quantifying such base-stacking perturbations.

The term “hybridized” refers to two nucleic acid strands associated witheach other which may or may not be fully base-paired: generally, thisterm refers to an association including an oligomer of the inventionwhether bound to a solid support or in solution.

The term “denaturing” refers to the process by which strands of nucleicacid duplexes (or higher order aggregates) are no longer base-paired byhydrogen bonding and are separated into single-stranded molecules.Methods of denaturation are well known to those skilled in the art andinclude thermal denaturation and alkaline denaturation. This termgenerally refers to the dissociation of a probe of the invention fromits target nucleic acid.

The term “mismatches” refers to nucleic acid nucleobases withinhybridized nucleic acid duplexes (or higher order aggregates) which arenot 100% complementary. A mismatch includes any incorrect pairingbetween the nucleobases of two nucleobases located on complementarystrands of nucleic acid that are not the Watson-Crick base-pairs, e.g.,A:T or G:C. The lack of total homology may be due to deletions,insertions, inversions, substitutions or frameshift mutations. Invarious embodiments, the oligomer of the invention includes a mismatchrelative to its target nucleic acid, preferably allowing detectionand/or characterization and/or quantification of the correspondingmismatch in its target. In certain embodiments, the mismatch is a singlenucleotide mismatch.

As used herein, the term “polymorphism” refers to a sequence variationin a gene, and “mutation” refers to a sequence variation in a gene thatis associated or believed to be associated with a phenotype. The term“gene” refers to a segment of the genome coding for a functional productprotein control region. Polymorphic markers used in accordance with thepresent invention for subject identification may be located in coding ornon-coding regions of the genome, and various probes of the inventionare designed to hybridize to nucleic acid regions including thesemarkers. The term “subject,” as used herein refers to a subjectproviding a test sample from which target nucleic acids are obtained forthe purpose of genetic testing. The oligomers of the invention are ofuse in detecting and/or characterizing and/or quantifying polymorphismsand mutations. Moreover, the solid supports of the invention are of usein synthesizing oligomers of use to detect and/or characterize and/orquantitate polymorphisms and mutations.

The term “probe” as used herein refers to nucleic acid oligomersprepared using a solid support or amidite of the invention. In variousembodiments, the probes produce a detectable response upon interactionwith a binding partner. The probes include at least one detectablemoiety, or a pair of moieties that form an energy transfer pairdetectable upon some change of state of the probe in response to itsinteraction with a binding partner. The present invention providesprobes and amidites and solid supports of use to synthesize probes.Exemplary probes of the invention are of use to detect a polymorphism.In various embodiments, the polymorphism is a single nucleic acidpolymorphism (SNP).

The term “detectable response” as used herein refers to a change in oran occurrence of, a signal that is directly or indirectly detectableeither by observation or by instrumentation and the presence of or,preferably, the magnitude of which is a function of the presence of atarget binding partner for a probe in the test sample. Typically, thedetectable response is an optical response from a fluorophore resultingin a change in the wavelength distribution patterns or intensity ofabsorbance or fluorescence or a change in light scatter, fluorescencequantum yield, fluorescence lifetime, fluorescence polarization, a shiftin excitation or emission wavelength or a combination of the aboveparameters. The detectable change in a given spectral property isgenerally an increase or a decrease. However, spectral changes thatresult in an enhancement of fluorescence intensity and/or a shift in thewavelength of fluorescence emission or excitation are also useful. Thechange in fluorescence on ion binding is usually due to conformationalor electronic changes in the indicator that may occur in either theexcited or ground state of the fluorophore, due to changes in electrondensity at the ion binding site, due to quenching of fluorescence by thebound target metal ion, or due to any combination of these or othereffects. Alternatively, the detectable response is an occurrence of asignal wherein the fluorophore is inherently fluorescent and does notproduce a change in signal upon binding to a metal ion or biologicalcompound. The present invention provides probes providing a detectableresponse and solid supports of use to synthesize such probes.

The term “carrier molecule” as used herein refers to any molecule towhich a compound of the invention is attached. Representative carriermolecules include a protein (e.g., enzyme, antibody), glycoprotein,peptide, saccharide (e.g., mono-, oligo-, and poly-saccharides),hormone, receptor, antigen, substrate, metabolite, transition stateanalog, cofactor, inhibitor, drug, dye, nutrient, growth factor, etc.,without limitation. “Carrier molecule” also refers to species that mightnot be considered to fall within the classical definition of “amolecule,” e.g., solid support (e.g., synthesis support, chromatographicsupport, membrane), virus and microorganism.

The symbol

, displayed perpendicular to a bond, indicates the point at which thedisplayed moiety is attached to the remainder of the molecule.

The compounds herein described may have one or more asymmetric centersor planes. Compounds of the present invention containing anasymmetrically substituted atom may be isolated in optically active orracemic forms. It is well known in the art how to prepare opticallyactive forms, such as by resolution of racemic forms (racemates), byasymmetric synthesis, or by synthesis from optically active startingmaterials. Resolution of the racemates can be accomplished, for example,by conventional methods such as crystallization in the presence of aresolving agent, or chromatography, using, for example a chiral HPLCcolumn. Many geometric isomers of olefins, C═N double bonds, and thelike can also be present in the compounds described herein, and all suchstable isomers are contemplated in the present invention. Cis and transgeometric isomers of the compounds of the present invention aredescribed and may be isolated as a mixture of isomers or as separatedisomeric forms. All chiral (enantiomeric and diastereomeric), andracemic forms, as well as all geometric isomeric forms of a structureare intended, unless the specific stereochemistry or isomeric form isspecifically indicated.

The graphic representations of racemic, ambiscalemic and scalemic orenantiomerically pure compounds used herein are taken from Maehr, J.Chem. Ed., 62: 114-120 (1985): solid and broken wedges are used todenote the absolute configuration of a chiral element; wavy linesindicate disavowal of any stereochemical implication which the bond itrepresents could generate; solid and broken bold lines are geometricdescriptors indicating the relative configuration shown but not implyingany absolute stereochemistry; and wedge outlines and dotted or brokenlines denote enantiomerically pure compounds of indeterminate absoluteconfiguration.

The term “charged group” refers to a group that bears a negative chargeor a positive charge. The negative charge or positive charge can have acharge number that is an integer selected from 1, 2, 3 or higher or thatis a fractional number. Exemplary charged groups include for example—OPO₃ ²⁻, —OPO₂ ⁻, —P⁺Ph₃, —N⁺R′R″R′″, —S⁺R and —C(O)O⁻.

The compounds herein described may have one or more charged groups. Forexample, the compounds may be zwitterionic, but may be neutral overall.Other embodiments may have one or more charged groups, depending on thepH and other factors. In these embodiments, the compound may beassociated with a suitable counter-ion. It is well known in the art howto prepare salts or exchange counter-ions. Generally, such salts can beprepared by reacting free acid forms of these compounds with astoichiometric amount of the appropriate base (such as Na, Ca, Mg, or Khydroxide, carbonate, bicarbonate, or the like), or by reacting freebase forms of these compounds with a stoichiometric amount of theappropriate acid. Such reactions are typically carried out in water orin an organic solvent, or in a mixture of the two. Counter-ions may bechanged, for example, by ion-exchange techniques such as ion-exchangechromatography. All zwitterions, salts and counter-ions are intended,unless the counter-ion or salt is specifically indicated. In certainembodiments, the salt or counter-ion may be pharmaceutically acceptable,for administration to a subject. Pharmaceutically acceptable salts arediscussed later.

Alternatively, the compounds of the invention may form a compoundincluding one or more charged groups following cleavage of the acidlabile moiety.

In some embodiments, the definition of terms used herein is according toIUPAC.

Dual Quencher Probes

In one aspect, the invention provides a nucleic acid probe comprising anoligonucleotide having attached thereto a first quencher and a secondquencher, wherein the first quencher is attached at an internal positionof the oligonucleotide.

The first quencher and second quencher are as defined herein.

In some embodiments, the oligonucleotide further comprises afluorophore. The fluorophore is as defined herein.

In some embodiments, the invention provides a nucleic acid probecomprising an oligonucleotide having the structure:5′-Y¹-L¹-L^(Q1)-L²-Y²-3′,

in which Y¹ comprises a sequence of two or more DNA or RNA nucleotides;Y² comprises a sequence of two or more DNA or RNA nucleotides. One of Y¹and Y² has a fluorophore covalently attached (directly or through alinker) to its nucleotide sequence, and the other of Y¹ and Y² has asecond quencher covalently attached (directly or through a linker) toits nucleotide sequence. L¹ and L² are independently selected from abond, substituted or unsubstituted alkyl, substituted or unsubstitutedcycloalkyl, substituted or unsubstituted heteroalkyl, and substituted orunsubstituted heterocycloalkyl. L^(Q1) is:

wherein Q¹ is a first quencher. The first quencher, second quencher andfluorophore are as defined herein.

Any of the combinations of Y¹, Y², L¹, L², and L^(Q1) are encompassed bythis disclosure and specifically provided by the invention.

In some embodiments, the nucleotide sequence of Y¹ includes a firstnucleotide N¹ having a 5′ phosphate covalently attached (directly orthrough a linker) to a fluorophore or a second quencher, and a secondnucleotide N² having a 3′ phosphate covalently attached to L¹.

In some embodiments, the nucleotide sequence of Y² includes a thirdnucleotide N³ having a 5′ phosphate covalently attached to L², and afourth nucleotide N⁴ having a 3′ phosphate covalently attached (directlyor through a linker) to a second quencher or a fluorophore.

In some embodiments, when the 5′ phosphate of the first nucleotide N¹ iscovalently attached (directly or through a linker) to the fluorophore,the 3′ phosphate of the fourth nucleotide N⁴ is covalently attached(directly or through a linker) to the second quencher; and when the 5′phosphate of the first nucleotide N¹ is covalently attached (directly orthrough a linker) to the second quencher, the 3′ phosphate of the fourthnucleotide N⁴ is covalently attached (directly or through a linker) tothe fluorophore.

In some embodiments, the nucleic acid probe further comprises one ormore DNA or RNA nucleotides attached to the linker that connects the 5′phosphate of the first nucleotide N¹ to the fluorophore or secondquencher. In some embodiments, the nucleic acid probe further comprisesone or more DNA or RNA nucleotides attached to the linker that connectsthe 3′ phosphate of the fourth nucleotide N⁴ to the second quencher orfluorophore.

In some embodiments, the oligonucleotide comprises from 15 to 45 (i.e.,15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32,33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, or 45) nucleotides. Insome embodiments, the oligonucleotide comprises from 20 to 30 (i.e., 20,21, 22, 23, 24, 25, 26, 27, 28, 29, or 30) nucleotides. In someembodiments, the oligonucleotide has from 15 to 45 (i.e., 15, 16, 17,18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35,36, 37, 38, 39, 40, 41, 42, 43, 44, or 45) nucleotides (total). In someembodiments, the oligonucleotide has from 20 to 30 (i.e., 20, 21, 22,23, 24, 25, 26, 27, 28, 29, or 30) nucleotides (total).

In some embodiments, the 5′ phosphate of the first nucleotide N¹ iscovalently attached (directly or through a linker) to the fluorophore;and the 3′ phosphate of the fourth nucleotide N⁴ is covalently attached(directly or through a linker) to the second quencher.

In some embodiments, when the 5′ phosphate of the first nucleotide N¹ iscovalently attached (directly or through a linker) to the fluorophore,Y¹ is a sequence of from 8 to 11 (i.e., 8, 9, 10, or 11) nucleotides. Insome embodiments, when the 5′ phosphate of the first nucleotide N¹ iscovalently attached (directly or through a linker) to the fluorophore,Y¹ is a sequence of 9 nucleotides.

In some embodiments, when the 5′ phosphate of the first nucleotide N¹ iscovalently attached (directly or through a linker) to the fluorophore,Y¹ is a sequence of from 8 to 11 nucleotides, and Y² is a sequence offrom 6 to 24 (i.e., 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,20, 21, 22, 23, or 24) nucleotides.

In some embodiments, the 5′ phosphate of the first nucleotide N¹ iscovalently attached (directly or through a linker) to the secondquencher; and the 3′ phosphate of the fourth nucleotide N⁴ is covalentlyattached (directly or through a linker) to the fluorophore.

In some embodiments, when the 3′ phosphate of the fourth nucleotide N⁴is covalently attached (directly or through a linker) to thefluorophore, Y² is a sequence of from 8 to 11 (i.e., 8, 9, 10, or 11)nucleotides. In some embodiments, when the 3′ phosphate of the fourthnucleotide N⁴ is covalently attached (directly or through a linker) tothe fluorophore, Y² is a sequence of 9 nucleotides.

In some embodiments, when the 3′ phosphate of the fourth nucleotide N⁴is covalently attached (directly or through a linker) to thefluorophore, Y² is a sequence of from 8 to 11 nucleotides, and Y¹ is asequence of from 6 to 24 (i.e., 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,17, 18, 19, 20, 21, 22, 23, or 24) nucleotides.

In some embodiments, L¹ is unsubstituted alkyl. In some embodiments, L¹is unsubstituted C₁-C₇ (i.e., C₁, C₂, C₃, C₄, C₅, C₆, or C₇) alkyl. Insome embodiments, L¹ is unsubstituted C₁-C₃ (i.e., C₁, C₂, or C₃) alkyl.In some embodiments, L¹ is methyl. In some embodiments, L¹ is ethyl. Insome embodiments, L¹ is n-propyl.

In some embodiments, L² is unsubstituted alkyl. In some embodiments, L²is unsubstituted C₁-C₇ (i.e., C₁, C₂, C₃, C₄, C₅, C₆, or C₇) alkyl. Insome embodiments, L² is unsubstituted C₁-C₃ (i.e., C₁, C₂, or C₃) alkyl.In some embodiments, L² is methyl. In some embodiments, L² is ethyl. Insome embodiments, L² is n-propyl.

In some embodiments, L¹ and L² are each the same. In some embodiments,L¹ and L² are different. In some embodiments, L¹ and L² are each methyl.In some embodiments, L¹ and L² are each ethyl. In some embodiments, L¹and L² are each n-propyl.

In some embodiments, the nucleic acid probe is an oligonucleotide havingthe structure:5′-Y¹-L¹-L^(Q1)-L²-Y²-3′wherein Y¹, Y², L¹, L², and L^(Q1) are as defined herein.QuenchersFirst (Internal) Quencher

In some embodiments, the first quencher (Q¹) has a structure comprisingat least three residues selected from substituted or unsubstituted aryl,substituted or unsubstituted heteroaryl, and combinations thereof,wherein at least two of the residues are covalently linked via anexocyclic diazo bond.

In some embodiments, each of the at least three residues is substitutedor unsubstituted phenyl.

In some embodiments, at least two of the at least three residues arephenyl substituted with at least one unsubstituted C₁-C₆ alkyl moiety.

In some embodiments, the first quencher (Q¹) is attached to theoligonucleotide through L³. L³ is as defined herein.

In some embodiments, the first quencher (Q¹) is attached to anucleotide, which is 8, 9, 10, or 11 nucleotides towards the 3′-terminusfrom the 5′-terminus of the oligonucleotide.

In some embodiments, the first quencher (Q¹) is attached between the 8thand 9th or the 9th and 10th or the 10th and 11th or the 11th and 12thnucleotides towards the 3′-terminus from the 5′-terminus of theoligonucleotide. In some embodiments, the first quencher (Q¹) isattached between the 9th and 10th nucleotides towards the 3′-terminusfrom the 5′-terminus of the oligonucleotide.

In some embodiments, the first quencher (Q¹) is attached to anucleotide, which is 8, 9, 10 or 11 nucleotides towards the 5′-terminusfrom the 3′-terminus of the oligonucleotide.

In some embodiments, the first quencher (Q¹) is attached between the 8thand 9th or the 9th and 10th or the 10th and 11th or the 11th and 12thnucleotides towards the 5′-terminus from the 3′-terminus of theoligonucleotide. In some embodiments, the first quencher (Q¹) isattached between the 9th and 10th nucleotides towards the 5′-terminusfrom the 3′-terminus of the oligonucleotide.

In some embodiments, the first quencher (Q¹) has a structure selectedfrom:

R^(a), R^(b), and R^(c) are independently selected from substituted orunsubstituted aryl. L³ is selected from a bond, substituted orunsubstituted alkyl, substituted or unsubstituted cycloalkyl,substituted or unsubstituted heteroalkyl, and substituted orunsubstituted heterocycloalkyl.

In some embodiments, R^(a), R^(b), and R^(c) are independently selectedfrom substituted or unsubstituted phenyl.

In some embodiments, L³ is a bond.

In some embodiments, the first quencher (Q¹) has a structure selectedfrom:

in which, R^(a2), R^(a3), R^(a5), R^(a6), R^(a7), R^(a8), R^(b2),R^(b3), R^(b4), R^(b5), R^(b6), R^(b7), R^(b8), R^(c2), R^(c3), R^(c4),R^(c5), R^(c6), R^(c7), and R^(c8) are independently selected from H,substituted or unsubstituted alkyl, substituted or unsubstituted alkoxy,and nitro. L³ is as defined herein.

In some embodiments, R^(a2), R^(a3), R^(a5), R^(a6), R^(a7), R^(a8),R^(b2), R^(b3), R^(b4), R^(b5), R^(b6), R^(b7), R^(b8), R^(c2), R^(c3),R^(c4), R^(c5), R^(c6), R^(c7), and R^(c8) are independently selectedfrom H, unsubstituted alkyl, and nitro.

In some embodiments, R^(a2), R^(a3), R^(a5), R^(a6), R^(a7), R^(a5),R^(b2), R^(b3), R^(b4), R^(b5), R^(b6), R^(b7), R^(b8), R^(c2), R^(c3),R^(c4), R^(c5), R^(c6), R^(c7), and R^(c8) are independently selectedfrom H, unsubstituted C₁-C₄ (i.e., C₁, C₂, C₃, or C₄) alkyl, and nitro.

In some embodiments, R^(a2), R^(a3), R^(a5), R^(a6), R^(a7), R^(a5),R^(b2), R^(b3), R^(b4), R^(b5), R^(b6), R^(b7), R^(b8), R^(c2), R^(c3),R^(c4), R^(c5), R^(c6), R^(c7), and R^(c8) are independently selectedfrom H, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl,sec-butyl, tert-butyl, and nitro.

In some embodiments, R^(a2), R^(a3), R^(a5), R^(a6), R^(a7) (ifpresent), and R^(a8) (if present) are H.

In some embodiments, R^(b2), R^(b3), b^(b4) (if present), R^(b5),R^(b6), b^(b7) (if present), and R^(b8) (if present) are independentlyselected from H, unsubstituted C₁-C₄ (i.e., C₁, C₂, C₃, or C₄) alkyl,and nitro. In some embodiments, R^(b2), R^(b3), R^(b5), R^(b6), R^(b7)(if present), and R^(b8) (if present) are independently selected from Hand unsubstituted C₁-C₄ (i.e., C₁, C₂, C₃, or C₄) alkyl. In someembodiments, R^(b4) is nitro.

In some embodiments, R^(c2), R^(c3), R^(c4), R^(c5), R^(c6), R^(c7) (ifpresent), and R^(c8) (if present) are independently selected from H,unsubstituted C₁-C₄ (i.e., C₁, C₂, C₃, or C₄) alkyl, and nitro. In someembodiments, R^(c2), R^(c3), R^(c5), R^(c6), R^(c7) (if present), andR^(c8) (if present) are independently selected from H and unsubstitutedC₁-C₄ (i.e., C₁, C₂, C₃, or C₄) alkyl.

Any of the combinations of R^(a2), R^(a3), R^(a5), R^(a6), R^(a7),R^(a8), R^(b2), R^(b3), R^(b4), R^(b5), R^(b6), R^(b7), R^(b8), R^(c2),R^(c3), R^(c4), R^(c5), R^(c6), R^(c7), R^(c8) and L³ are encompassed bythis disclosure and specifically provided by the invention.

In some embodiments, Q¹ has the structure:

wherein R^(a2), R^(a3), R^(a5), and R^(a6) are H; and R^(b2) R^(b3),R^(b5), R^(b6), R^(c2), R^(c3), R^(c4), R^(c5), R^(c6) and L³ are asdefined herein.

In some embodiments, at least one (i.e., one, two, three, or all) ofR^(b2), R^(b3), R^(b5), and R^(b6) is independently methyl or ethyl orn-propyl or isopropyl or n-butyl or isobutyl or sec-butyl or tert-butyl,and the remainder of R^(b2) R^(b3) R^(b5) and R^(b6) are H. In someembodiments, one of R^(b2), R^(b3), R^(b5), and R^(b6) is methyl orethyl or n-propyl or isopropyl or n-butyl or isobutyl or sec-butyl ortert-butyl, and the remainder of R^(b2), R^(b3), R^(b5), and R^(b6) areH.

In some embodiments, at least one (i.e., one, two, three, four, or all)of R^(c2), R^(c3), R^(c4), R^(c5), and R^(c6) is independently methyl orethyl or n-propyl or isopropyl or n-butyl or isobutyl or sec-butyl ortert-butyl, and the remainder of R^(c2), R^(c3), R^(c4), R^(c5), andR^(c6) are H. In some embodiments, one of R^(c2), R^(c3), R^(c4),R^(c5), and R^(c6) is methyl or ethyl or n-propyl or isopropyl orn-butyl or isobutyl or sec-butyl or tert-butyl, and the remainder ofR^(c2), R^(c3), R^(c4), R^(c5), and R^(c6) are H.

In some embodiments, the first quencher (Q¹) has the structure:

wherein R^(b2), R^(c2), R^(c4) and L³ are as defined herein.

In some embodiments, R^(c4) is H. In some embodiments, R^(b2) and R^(c2)are independently methyl or ethyl or n-propyl or isopropyl or n-butyl orisobutyl or sec-butyl or tert-butyl; and R^(c4) is H.

In some embodiments, the first quencher (Q¹) has a structure selectedfrom:

wherein L³ is as defined herein.

In some embodiments, the first quencher (Q¹) has the structure:

Second Quencher

In some embodiments, the second quencher has a structure comprising atleast three residues selected from substituted or unsubstituted aryl,substituted or unsubstituted heteroaryl, and combinations thereof,wherein at least two of the residues are covalently linked via anexocyclic diazo bond.

In some embodiments, the second quencher is attached to theoligonucleotide through L⁴. L⁴ is as defined herein.

In some embodiments, the second quencher has the structure:

wherein R^(e2), R^(e5), R^(f2), and R^(f4) are independently selectedfrom H, substituted or unsubstituted alkyl, substituted or unsubstitutedalkoxy, and nitro; and L⁴ is selected from a bond, substituted orunsubstituted alkyl, substituted or unsubstituted cycloalkyl,substituted or unsubstituted heteroalkyl, and substituted orunsubstituted heterocycloalkyl.

In some embodiments, R^(e2) and R^(e5) are independently selected fromH, unsubstituted alkyl, and unsubstituted alkoxy. In some embodiments,R^(e2) and R^(e5) are independently selected from H, unsubstituted C₁-C₄(i.e., C₁, C₂, C₃, or C₄) alkyl, and unsubstituted C₁-C₄ (i.e., C₁, C₂,C₃, or C₄) alkoxy. In some embodiments, R^(e2) and R^(e5) areindependently selected from H, methyl, and methoxy.

In some embodiments, R^(f2) and R^(f4) are independently selected fromH, unsubstituted alkyl, and nitro. In some embodiments, R^(f2) andR^(f4) are independently selected from H, unsubstituted C₁-C₄ (i.e., C₁,C₂, C₃, or C₄) alkyl, and nitro. In some embodiments, R^(f2) and R^(f4)are independently selected from H, methyl, and nitro.

In some embodiments, R^(e2) is unsubstituted C₁-C₄ alkyl; R^(e5) is H;R^(f2) is unsubstituted C₁-C₄ alkyl; and R^(f4) is H.

In some embodiments, R^(e2) is unsubstituted C₁-C₄ alkoxy; R^(e5) isunsubstituted C₁-C₄ alkyl; R^(f2) is nitro; and R^(f4) is unsubstitutedC₁-C₄ alkyl.

In some embodiments, R^(e2) is unsubstituted C₁-C₄ alkoxy; R^(e5) isunsubstituted C₁-C₄ alkoxy; R^(f2) is H; and R^(f4) is nitro.

In some embodiments, L⁴ is a substituted or unsubstituted dialkylamine.

In some embodiments, L⁴ has the structure:

wherein L^(N) is selected from substituted or unsubstituted alkyl andsubstituted or unsubstituted heteroalkyl; and R^(N) is selected from H,substituted or unsubstituted alkyl, and substituted or unsubstitutedheteroalkyl.

In some embodiments, the attachment point at the nitrogen atom shown inthe formula above is the attachment point to the phenyl moiety of thesecond quencher. In some embodiments, L^(N) is unsubstituted alkyl. Insome embodiments, L^(N) is unsubstituted C₁-C₄ (i.e., C₁, C₂, C₃, or C₄)alkyl. In some embodiments, L^(N) is ethyl.

In some embodiments, R^(N) is substituted or unsubstituted alkyl. Insome embodiments, R^(N) is substituted or unsubstituted C₁-C₄ (i.e., C₁,C₂, C₃, or C₄) alkyl. In some embodiments, R^(N) is unsubstituted C₁-C₄(i.e., C₁, C₂, C₃, or C₄) hydroxyalkyl. In some embodiments, R^(N) isethyl. In some embodiments, R^(N) is 2-hydroxyethyl.

In some embodiments, the second quencher has the structure:

wherein R^(e2), R^(e5), R^(f2), R^(f4), L^(N) and R^(N) are as definedherein.

In some embodiments, the second quencher has the structure:

wherein L⁴ is as defined herein.

In some embodiments, the second quencher has the structure:

The second quencher can be derived from a quencher reagent, for examplethrough reaction of a reactive functional group on the quencher reagentwith a reactive functional group of complementary reactivity on theoligonucleotide or on a linker attached to the oligonucleotide.

In some embodiments, the second quencher is (derived from) a BHQ (BlackHole Quencher®; Biosearch Technologies, Inc.), such as a quencherdescribed in U.S. Pat. No. 7,019,129. In some embodiments, the secondquencher is a BHQ-0, BHQ-1, BHQ-2, or BHQ-3 derivative having thestructure:

wherein L⁴ is as defined herein.

In some embodiments, the second quencher is (derived from) a quencherdescribed in U.S. Provisional Patent Application No. 61/990,913; filedMay 9, 2014; titled “Cosmic Quenchers.” In some embodiments, the secondquencher has the structure:

wherein L⁴ is as defined herein.

In some embodiments, the second quencher is (derived from) a BlackBerryQuencher (BBQ-650®; Berry & Associates, Inc.; described in U.S. Pat. No.7,879,986). In some embodiments, the second quencher has the structure:

wherein L⁴ is as defined herein.

In some embodiments, the structure of the second quencher is any of thestructures shown herein for the first quencher (Q′).

In some embodiments, the second quencher is (derived from) Dabcyl,Dabsyl, Eclipse® quencher, Iowa Black® FQ, Iowa Black® RQ-n1, or IowaBlack® RQ-n2.

In some embodiments, the second quencher is attached to the 3′- or the5′-terminus of the oligonucleotide. In some embodiments, the secondquencher is attached to the 3′-terminus of the oligonucleotide.

In some embodiments, the second quencher is attached to the 3′ phosphateof the fourth nucleotide N⁴ or to the 5′ phosphate of the firstnucleotide N¹. In some embodiments, the second quencher is attached tothe 3′ phosphate of the fourth nucleotide N⁴.

Fluorophore

One of the advantages of the compounds of the invention is that a widerange of energy donor molecules can be used in conjunction with thequencher-functionalized nucleic acid probes and oligonucleotides. A vastarray of fluorophores is known to those of skill in the art. See, forexample, Cardullo et al., Proc. Natl. Acad. Sci. USA 85: 8790-8794(1988); Dexter, D. L., J. of Chemical Physics 21: 836-850 (1953);Hochstrasser et al., Biophysical Chemistry 45: 133-141 (1992); Selvin,P., Methods in Enzymology 246: 300-334 (1995); Steinberg, I. Ann. Rev.Biochem., 40: 83-114 (1971); Stryer, L. Ann. Rev. Biochem., 47: 819-846(1978); Wang et al., Tetrahedron Letters 31: 6493-6496 (1990); Wang etal., Anal. Chem. 67: 1197-1203 (1995).

A non-limiting list of exemplary donors that can be used in conjunctionwith the quenchers of the invention is provided in Table 1.

TABLE 1 Suitable moieties that can be selected as donors or acceptors indonor-acceptor energy transfer pairs4-acetamido-4′-isothiocyanatostilbene-2,2′disulfonic acid acridine andderivatives: acridine acridine isothiocyanate5-(2′-aminoethyl)aminonaphthalene-1-sulfonic acid (EDANS)4-amino-N-[3-vinylsulfonyl)phenyl]naphthalimide-3,5 disulfonateN-(4-anilino-1-naphthyl)maleimide anthranilamide BODIPY Brilliant Yellowcoumarin and derivatives: coumarin 7-amino-4-methylcoumarin (AMC,Coumarin 120) 7-amino-4-trifluoromethylcouluarin (Coumaran 151) cyaninedyes cyanosine 4′,6-diaminidino-2-phenylindole (DAPI)5′,5″-dibromopyrogallol-sulfonaphthalein (Bromopyrogallol Red)7-diethylamino-3-(4′-isothiocyanatophenyl)-4-methylcoumarindiethylenetriamine pentaacetate4,4′-diisothiocyanatodihydro-stilbene-2,2′-disulfonic acid4,4′-diisothiocyanatostilbene-2,2′-disulfonic acid5-[dimethylamino]naphthalene-1-sulfonyl chloride (DNS, dansylchloride)4-(4′-dimethylaminophenylazo)benzoic acid (DABCYL)4-dimethylaminophenylazophenyl-4′-isothiocyanate (DABITC) eosin andderivatives: eosin eosin isothiocyanate erythrosin and derivatives:erythrosin B erythrosin isothiocyanate ethidium fluorescein andderivatives: 5-carboxyfluorescein (FAM)5-(4,6-dichlorotriazin-2-yl)aminofluorescein (DTAF)2′,7′-dimethoxy-4′5′-dichloro-6-carboxyfluorescein (JOE) fluoresceinfluorescein isothiocyanate QFITC (XRITC) fluorescamine IR144 IR1446Malachite Green isothiocyanate 4-methylumbelliferone orthocresolphthalein nitrotyrosine pararosaniline Phenol Red B-phycoerythrino-phthaldialdehyde pyrene and derivatives: pyrene pyrene butyratesuccinimidyl 1-pyrene butyrate quantum dots Reactive Red 4 (Cibacron ™Brilliant Red 3B-A) rhodamine and derivatives: 6-carboxy-X-rhodamine(ROX) 6-carboxyrhodamine (R6G) lissamine rhodamine B sulfonyl chloriderhodamine (Rhod) rhodamine B rhodamine 123 rhodamine X isothiocyanatesulforhodamine B sulforhodamine 101 sulfonyl chloride derivative ofsulforhodamine 101 (Texas Red) N,N,N′,N′-tetramethyl-6-carboxyrhodamine(TAMRA) tetramethyl rhodamine tetramethyl rhodamine isothiocyanate(TRITC) riboflavin rosolic acid metal chelates, e.g., lanthanidechelates (e.g., europium terbium chelates), ruthenium chlelates

There is a great deal of practical guidance available in the literaturefor selecting appropriate donor-acceptor pairs for particular probes, asexemplified by the following references: Pesce et al., Eds.,FLUORESCENCE SPECTROSCOPY (Marcel Dekker, New York, 1971); White et al.,FLUORESCENCE ANALYSIS: A PRACTICAL APPROACH (Marcel Dekker, New York,1970); and the like. The literature also includes references providingexhaustive lists of fluorescent and chromogenic molecules and theirrelevant optical properties for choosing reporter-quencher pairs (see,for example, Berlman, HANDBOOK OF FLUORESCENCE SPECTRA OF AROMATICMOLECULES, 2nd Edition (Academic Press, New York, 1971); Griffiths,COLOUR AND CONSTITUTION OF ORGANIC MOLECULES (Academic Press, New York,1976); Bishop, Ed., INDICATORS (Pergamon Press, Oxford, 1972); Haugland,HANDBOOK OF FLUORESCENT PROBES AND RESEARCH CHEMICALS (Molecular Probes,Eugene, 1992) Pringsheim, FLUORESCENCE AND PHOSPHORESCENCE (IntersciencePublishers, New York, 1949); and the like. Further, there is extensiveguidance in the literature for derivatizing reporter and quenchermolecules for covalent attachment via common reactive groups that can beadded to a nucleic acid, as exemplified by the following references:Haugland (supra); Ullman et al., U.S. Pat. No. 3,996,345; Khanna et al.,U.S. Pat. No. 4,351,760. Thus, it is well within the abilities of thoseof skill in the art to choose an energy exchange pair for a particularapplication and to conjugate the members of this pair to a probemolecule, such as, for example, a nucleic acid, peptide or otherpolymer.

Generally, it is preferred that an absorbance band of the quenchersubstantially overlap the fluorescence emission band of the donor. Whenthe donor (fluorophore) is a component of a probe that utilizesdonor-acceptor energy transfer, the donor fluorescent moiety and thequencher (acceptor) of the invention are preferably selected so that thedonor and acceptor moieties exhibit donor-acceptor energy transfer whenthe donor moiety is excited. One factor to be considered in choosing thefluorophore-quencher pair is the efficiency of donor-acceptor energytransfer between them. Preferably, the efficiency of FRET between thedonor and acceptor moieties is at least 10%, more preferably at least50% and even more preferably at least 80%. The efficiency of FRET caneasily be empirically tested using the methods both described herein andknown in the art.

The efficiency of energy transfer between the donor-acceptor pair canalso be adjusted by changing the ability of the donor and acceptorgroups to dimerize or closely associate. If the donor and acceptormoieties are known or determined to closely associate, an increase ordecrease in association can be promoted by adjusting the length of alinker moiety, or of the probe itself, between the donor and acceptor.The ability of donor-acceptor pair to associate can be increased ordecreased by tuning the hydrophobic or ionic interactions, or the stericrepulsions in the probe construct. Thus, intramolecular interactionsresponsible for the association of the donor-acceptor pair can beenhanced or attenuated. Thus, for example, the association between thedonor-acceptor pair can be increased by, for example, utilizing a donorbearing an overall negative charge and an acceptor with an overallpositive charge.

In addition to fluorophores that are attached directly to a probe, thefluorophores can also be attached by indirect means. In this embodiment,a ligand molecule (e.g., biotin) is generally covalently bound to theprobe species. The ligand then binds to another molecules (e.g.,streptavidin) molecule, which is either inherently detectable orcovalently bound to a signal system, such as a fluorescent compound, oran enzyme that produces a fluorescent compound by conversion of anon-fluorescent compound. Useful enzymes of interest as labels include,for example, hydrolases, particularly phosphatases, esterases andglycosidases, hydrolases, peptidases or oxidases, particularlyperoxidases, and Fluorescent compounds include fluorescein and itsderivatives, rhodamine and its derivatives, dansyl, umbelliferone, etc.,as discussed above. For a review of various labeling or signal producingsystems that can be used, see, U.S. Pat. No. 4,391,904.

Donors of use in conjunction with the quenchers of the invention,include, for example, xanthene dyes, including fluoresceins, cyaninedyes and rhodamine dyes. Many suitable forms of these compounds arewidely available commercially with substituents on their phenylmoieties, which can be used as the site for bonding or as the bondingfunctionality for attachment to an nucleic acid. Another group offluorescent compounds of use in conjunction with the quenchers of theinvention are the naphthylamines, having an amino group in the alpha orbeta position. Included among such naphthylamino compounds are1-dimethylaminonaphthyl-5-sulfonate, 1-anilino-8-naphthalene sulfonateand 2-p-touidinyl-6-naphthalene sulfonate. Other donors include3-phenyl-7-isocyanatocoumarin, acridines, such as9-isothiocyanatoacridine and acridine orange;N-(p-(2-benzoxazolyl)phenyl)maleimide; benzoxadiazoles, stilbenes,pyrenes, and the like.

In an exemplary embodiment, in which the probe is a nucleic acid probe,the fluorophore is a fluorescein (e.g., FAM). The fluorophore ispreferably attached to either the 3′- or the 5′-terminus of the nucleicacid, although internal sites are also accessible and have utility forselected purposes. Whichever terminus the fluorophore is attached to,the quencher will generally be attached to its antipode, or at aposition internal to the nucleic acid chain. Donor groups are preferablyintroduced using an amidite derivative of the donor. Alternatively,donor groups comprising reactive functional groups (e.g.,isothiocyanates, active esters, etc.) can be introduced via reactionwith a reactive functional group on a tether or linker arm attached tothe nucleic acid (e.g., hexyl amine).

In yet another preferred embodiment, the donor moiety can be attached atthe 3′-terminus of a nucleic acid by the use of a derivatized synthesissupport. For example, TAMRA (tetramethylrhodamine carboxylic acid) isattached to a nucleic acid 3′-terminus using a solid support that isderivatized with an analogue of this fluorophore (BiosearchTechnologies, Inc.)

In view of the well-developed body of literature concerning theconjugation of small molecules to nucleic acids, many other methods ofattaching donor/acceptor pairs to nucleic acids will be apparent tothose of skill in the art. For example, rhodamine and fluorescein dyesare conveniently attached to the 5′-hydroxyl of a nucleic acid at theconclusion of solid phase synthesis by way of dyes derivatized with aphosphoramidite moiety (see, for example, Woo et al., U.S. Pat. No.5,231,191; and Hobbs, Jr., U.S. Pat. No. 4,997,928).

More specifically, there are many linker moieties and methodologies forattaching groups to the 5′- or 3′-termini of nucleic acids, asexemplified by the following references: Eckstein, editor, Nucleic acidsand Analogues: A Practical Approach (IRL Press, Oxford, 1991); Zuckermanet al., Nucleic Acids Research, 15: 5305-5321 (1987) (3′-thiol group onnucleic acid); Sharma et al., Nucleic Acids Research, 19: 3019 (1991)(3′-sulfhydryl); Giusti et al., PCR Methods and Applications, 2: 223-227(1993) and Fung et al., U.S. Pat. No. 4,757,141 (5′-phosphoamino groupvia Aminolink™ II available from P.E. Biosystems, CA.) Stabinsky, U.S.Pat. No. 4,739,044 (3-aminoalkylphosphoryl group); Agrawal et al.,Tetrahedron Letters, 31: 1543-1546 (1990) (attachment viaphosphoramidate linkages); Sproat et al., Nucleic Acids Research, 15:4837 (1987) (5-mercapto group); Nelson et al., Nucleic Acids Research,17: 7187-7194 (1989) (3′-amino group), and the like.

Means of detecting fluorescent labels are well known to those of skillin the art. Thus, for example, fluorescent labels can be detected byexciting the fluorophore with the appropriate wavelength of light anddetecting the resulting fluorescence. The fluorescence can be detectedvisually, by means of photographic film, by the use of electronicdetectors such as charge coupled devices (CCDs) or photomultipliers andthe like. Similarly, enzymatic labels may be detected by providing theappropriate substrates for the enzyme and detecting the resultingreaction product.

The fluorophore can be a non-protein organic fluorophore or afluorescent protein. Exemplary families of non-protein organicfluorophores are: Xanthene derivatives (such as fluorescein, rhodamine,Oregon green, eosin, and Texas red), cyanine derivatives (such ascyanine, indocarbocyanine, oxacarbocyanine, thiacarbocyanine, andmerocyanine), squaraine derivatives and ring-substituted squaraines(including Seta, SeTau, and Square dyes), naphthalene derivatives (suchas dansyl and prodan derivatives), coumarin derivatives, oxadiazolederivatives (such as pyridyloxazole, nitrobenzoxadiazole andbenzoxadiazole), anthracene derivatives (such as anthraquinones,including DRAQ5, DRAQ7 and CyTRAK Orange), pyrene derivatives (such ascascade blue), oxazine derivatives (such as Nile red, Nile blue, cresylviolet, and oxazine 170), acridine derivatives (such as proflavin,acridine orange, and acridine yellow), arylmethine derivatives (such asauramine, crystal violet, and malachite green), and tetrapyrrolederivatives (such as porphin, phthalocyanine, and bilirubin).

In some embodiments, the fluorophore is (derived from)6-carboxyfluorescein (FAM),2′7′-dimethoxy-4′5′-dichloro-6-carboxyfluorescein (JOE),tetrachlorofluorescein (TET), 6-carboxyrhodamine (R6G),N,N,N′,N′-tetramethyl-6-carboxyrhodamine (TAMRA), 6-carboxy-X-rhodamine(ROX), 1-dimethylaminonaphthyl-5-sulfonate, 1-anilino-8-naphthalenesulfonate, 2-p-toluidinyl-6-naphthalene sulfonate,5-(2′-aminoethyl)aminonaphthalene-1-sulfonic acid (EDANS), a coumarindye, an acridine dye, indodicarbocyanine 3 (Cy3), indodicarbocyanine 5(Cy5), indodicarbocyanine 5.5 (Cy5.5),3-(1-carboxy-pentyl)-3′-ethyl-5,5′-dimethyloxacarbocyanine (CyA),1H,5H,11H,15H-Xantheno[2,3,4-ij:5,6,7-i′j′]diquinolizin-18-ium, 9-[2(or4)-[[[6-[2,5-dioxo-1-pyrrolidinyl)oxy]-6-oxohexyl]amino]sulfonyl]-4(or2)-sulfophenyl]-2,3,6,7,12,13,16,17-octahydro-inner salt (TR or TexasRed), a BODIPY dye, benzoxaazole, stilbene, or pyrene.

In some embodiments, the fluorophore is derived from6-carboxyfluorescein (FAM). In some embodiments, the fluorophorecomprises a fluorescein moiety.

In some embodiments, the fluorophore is attached to the oligonucleotidethrough L⁵. L⁵ is as defined herein.

In some embodiments, the fluorophore has the structure:

wherein L⁵ is selected from a bond, substituted or unsubstituted alkyl,substituted or unsubstituted cycloalkyl, substituted or unsubstitutedheteroalkyl, and substituted or unsubstituted heterocycloalkyl.

In some embodiments, L⁵ is selected from a bond, substituted orunsubstituted alkyl, and substituted or unsubstituted heteroalkyl.

In some embodiments, the fluorophore has the structure:

In some embodiments, the fluorophore is derived from6-carboxy-tetrachlorofluorescein. In some embodiments, the fluorophorecomprises a tetrachlorofluorescein (TET) moiety.

In some embodiments, the fluorophore is attached to the oligonucleotidethrough L⁵. L⁵ is as defined herein.

In some embodiments, the fluorophore has the structure:

wherein L⁵ is selected from a bond, substituted or unsubstituted alkyl,substituted or unsubstituted cycloalkyl, substituted or unsubstitutedheteroalkyl, and substituted or unsubstituted heterocycloalkyl.

In some embodiments, L⁵ is selected from a bond, substituted orunsubstituted alkyl, and substituted or unsubstituted heteroalkyl.

In some embodiments, the fluorophore has the structure:

In some embodiments, the fluorophore is (derived from) a xanthene dyedescribed in U.S. Pat. No. 7,344,701.

In some embodiments, the fluorophore is (derived from) CAL Fluor® Gold540 (LGC Biosearch Technologies). In some embodiments, the fluorophorecomprises a CAL Fluor® Gold 540 (LGC Biosearch Technologies) moiety.

In some embodiments, the fluorophore is attached to the oligonucleotidethrough L⁵. L⁵ is as defined herein.

In some embodiments, the fluorophore has the structure:

wherein L⁵ is selected from a bond, substituted or unsubstituted alkyl,substituted or unsubstituted cycloalkyl, substituted or unsubstitutedheteroalkyl, and substituted or unsubstituted heterocycloalkyl.

In some embodiments, L⁵ is selected from a bond, substituted orunsubstituted alkyl, and substituted or unsubstituted heteroalkyl.

In some embodiments, the fluorophore has the structure:

In some embodiments, the fluorophore is (derived from) CAL Fluor® Orange560 (LGC Biosearch Technologies). In some embodiments, the fluorophorecomprises a CAL Fluor® Orange 560 (LGC Biosearch Technologies) moiety.

In some embodiments, the fluorophore is attached to the oligonucleotidethrough L⁵. L⁵ is as defined herein.

In some embodiments, the fluorophore has the structure:

wherein L⁵ is selected from a bond, substituted or unsubstituted alkyl,substituted or unsubstituted cycloalkyl, substituted or unsubstitutedheteroalkyl, and substituted or unsubstituted heterocycloalkyl.

In some embodiments, L⁵ is selected from a bond, substituted orunsubstituted alkyl, and substituted or unsubstituted heteroalkyl.

In some embodiments, the fluorophore has the structure:

In some embodiments, the fluorophore is derived from6-carboxy-2′,4,4′,5′,7,7′-hexachlorofluorescein (HEX). In someembodiments, the fluorophore comprises a6-carboxy-2′,4,4′,5′,7,7′-hexachlorofluorescein (HEX) moiety.

In some embodiments, the fluorophore is attached to the oligonucleotidethrough L⁵. L⁵ is as defined herein.

In some embodiments, the fluorophore has the structure:

wherein L⁵ is selected from a bond, substituted or unsubstituted alkyl,substituted or unsubstituted cycloalkyl, substituted or unsubstitutedheteroalkyl, and substituted or unsubstituted heterocycloalkyl.

In some embodiments, L⁵ is selected from a bond, substituted orunsubstituted alkyl, and substituted or unsubstituted heteroalkyl.

In some embodiments, the fluorophore has the structure:

In some embodiments, the fluorophore has a structure selected from:

L⁵ is as defined herein.

In some embodiments, the fluorophore has a structure selected from:

In some embodiments, the fluorophore is attached to the 5′- or the3′-terminus of the oligonucleotide. In some embodiments, the fluorophoreis attached to the 5′-terminus of the oligonucleotide. In someembodiments, the fluorophore is attached to the 5′ phosphate of thefirst nucleotide N¹ or to the 3′ phosphate of the fourth nucleotide N⁴.In some embodiments, the fluorophore is attached to the 5′ phosphate ofthe first nucleotide N¹.

Exemplary Quencher/Fluorophore Combinations

Any of the combinations of the first quencher (Q¹), the second quencher,and the fluorophore as defined herein are encompassed by this disclosureand specifically provided by the invention.

In some embodiments, the first quencher (Q¹) has the structure:

the second quencher has the structure:

and the fluorophore has a structure selected from:

L³, L⁴, and L⁵ are as defined herein.

In some embodiments, the first quencher (Q¹) has the structure:

the second quencher has the structure:

and the fluorophore has a structure selected from:

In some embodiments, the first quencher (Q¹) has the structure:

the second quencher has the structure:

and the fluorophore has the structure:

Oligomers

Also provided is a nucleic acid oligomer, e.g., a probe. Exemplaryoligomers include two quenchers (i.e., a first and a second quencher)covalently attached thereto, each optionally through a linker. Exemplaryoligomers include two quenchers (i.e., a first and a second quencher)and a fluorophore covalently attached thereto, each optionally through alinker.

Exemplary oligomers include oligonucleotides, oligonucleosides,oligodeoxyribonucleotides (containing 2′-deoxy-D-ribose or modifiedforms thereof), i.e.,

DNA, oligoribonucleotides (containing D-ribose or modified formsthereof), i.e., RNA, and any other type of polynucleotide which is anN-glycoside or C-glycoside of a purine or pyrimidine nucleobase, ormodified purine or pyrimidine nucleobase. Oligomer as used herein alsoincludes compounds where adjacent nucleomonomers are linked via amidelinkages as previously described (Nielsen et al., Science (1991)254:1497-1500). Elements ordinarily found in oligomers, such as thefuranose ring and/or the phosphodiester linkage can be replaced with anysuitable functionally equivalent element. “Oligomer” is thus intended toinclude any structure that serves as a scaffold or support for thenucleobases wherein the scaffold permits binding to target nucleic acidsin a sequence-dependent manner.

Exemplary groups linking nucleomonomers in an oligomer of the inventioninclude (i) phosphodiester and phosphodiester modifications(phosphorothioate, methylphosphonate, etc), (ii) substitute linkagesthat contain a non-phosphorous isostere (formacetal, riboacetal,carbamate, etc), (iii) morpholino residues, carbocyclic residues orother furanose sugars, such as arabinose, or a hexose in place of riboseor deoxyribose and (iv) nucleomonomers linked via amide bonds or acyclicnucleomonomers linked via any suitable substitute linkage.

The oligomers of the invention can be formed using modified andconventional nucleomonomers and synthesized using standard solid phase(or solution phase) oligomer synthesis techniques, which are nowcommercially available. In general, the oligomers can be synthesized bya method comprising the steps of: synthesizing a nucleomonomer oroligomer synthon having a protecting group and a nucleobase and acoupling group capable of coupling to a nucleomonomer or oligomer;coupling the nucleomonomer or oligomer synthon to an acceptornucleomonomer or an acceptor oligomer; removing the protecting group;and repeating the cycle as needed until the desired oligomer issynthesized.

The oligomers of the present invention can be of any length includingthose of greater than 40, 50 or 100 nucleomonomers. In variousembodiments, oligomers contain 2-100 nucleomonomers. Lengths of greaterthan or equal to about 10 to 40 nucleomonomers are useful fortherapeutic or diagnostic applications. Short oligomers containing 2, 3,4 or 5 nucleomonomers are specifically included in the present inventionand are useful, e.g., as synthons.

Oligomers having a randomized sequence and containing fewer than 20,fewer than 15 or fewer than 10 nucleomonomers are useful for primers,e.g., in cloning or amplification protocols that use random sequenceprimers, provided that the oligomer contains residues that can serve asa primer for polymerases or reverse transcriptases.

Oligomers can contain conventional phosphodiester linkages or cancontain phosphodiester modification such as phosphoramidate linkages.These substitute linkages include, but are not limited to, embodimentswherein a moiety of the formula —O—P(O)(S)—O—(“Phosphorothioate”),—O—P(S)(S)—O— (“phosphorodithioate”), —O—P(O)— (NR^(o) ₂)—X—,))—O—P(O)(R^(o))—O—O—P(S)(R^(o)—O— (“thionoalkylphosphonate”),—P(O)(OR^(p))—X—, —O—C(O)—X—, or —O—C(O)(NR^(p) ₂)—X—, wherein R^(o) isH (or a salt) or alkyl (C₁-C₁₂) and R^(p) is alkyl (C₁-C₉) and thelinkage is joined to adjacent nucleomonomers through an —O— or —S—bonded to a carbon of the nucleomonomer. In various embodiments, thesubstitute linkages for use in the oligomers of the present inventioninclude phosphodiester, phosphorothioate, methylphosphonate andthionomethylphosphonate linkages. Phosphorothioate and methylphosphonatelinkages confer added stability to the oligomer in physiologicalenvironments. While not all such linkages in the same oligomer need beidentical, particularly preferred oligomers of the invention containuniformly phosphorothioate linkages or uniformly methylphosphonatelinkages.

Oligomers or the segments thereof are conventionally synthesized, andcan be prepared using a compound of the invention. The synthetic methodsknown in the art and described herein can be used to synthesizeoligomers containing compounds of the invention, as well as othernucleobases known in the art, using appropriately protectednucleomonomers. Methods for the synthesis of oligomers are found, forexample, in Froehler, B., et al., Nucleic Acids Res. (1986)14:5399-5467; Nucleic Acids Res. (1988) 16:4831-4839; Nucleosides andNucleotides (1987) 6:287-291; Froehler, B., Tetrahedron Lett. (1986)27:5575-5578; Caruthers, M. H. in Oligodeoxynucleotides-AntisenseInhibitions of Gene Expression (1989), J. S. Cohen, editor, CRC Press,Boca Raton, p7-24; Reese, C. B. et al., Tetrahedron Lett. (1985)26:2245-2248. Synthesis of the methylphosphonate linked oligomers viamethyl phosphonamidite chemistry has also been described (Agrawal, S. etal., Tetrahedron Lett. (1987) 28:3539-3542; Klem, R. E., et al.,International Publication Number WO 92/07864).

As disclosed herein, the invention provides “conjugates” of oligomers.For instance, the oligomers can be covalently linked to variousfunctional components such as, stabilizing moieties, fluorophores,quenchers, intercalators, and substances which interact specificallywith the minor groove of the DNA double helix (minor groove binders,“MGB”). Other chosen conjugate moieties, can be labels such asradioactive, fluorescent, enzyme, or moieties which facilitate cellassociation using cleavage linkers and the like. Suitable radiolabelsinclude ³²P, ³⁵S, ³H and ¹⁴C; and suitable fluorescent labels includefluorescein, resorufin, rhodamine, BODIPY (Molecular Probes) and texasred; suitable enzymes include alkaline phosphatase and horseradishperoxidase. Additional fluorophores are set forth herein and aregenerally recognized in the art. Other covalently linked moietiesinclude biotin, antibodies or antibody fragments, and proteins, e.g.,transferrin and the HIV Tat protein.

As discussed herein and recognized in the art, the oligomers can bederivatized through any convenient linkage. For example, minor groovebinders, fluorophores, quenchers and intercalators, such as acridine orpsoralen can be linked to the oligomers of the invention through anyavailable —OH or —SH, e.g., at the terminal 5′-position of the oligomer,the 2′-positions of RNA, or an OH, NH₂, COOH or SH incorporated into the5-position of pyrimidines. A derivatized form which contains, forexample, —CH₂CH₂NH₂, —CH₂CH₂CH₂OH or —CH₂CH₂CH₂SH in the 5-position isof use in the present invention. Conjugates including polylysine orlysine can be synthesized as described and can further enhance thebinding affinity of an oligomer to its target nucleic acid sequence(Lemaitre, M. et al., Proc Natl Acad Sci (1987) 84:648-652; Lemaitre, M.et al., Nucleosides and Nucleotides (1987) 6:311-315).

A wide variety of substituents can be attached, including those boundthrough linkages or substitute linkages. The —OH moieties in thephosphodiester linkages of the oligomers can be replaced by phosphategroups, protected by standard protecting groups, or coupling groups toprepare additional linkages to other nucleomonomers, or can be bound tothe conjugated substituent. The 5′-terminal OH can be phosphorylated;the 2′-OH or OH substituents at the 3′-terminus can also bephosphorylated. The hydroxyls can also be derivatized to standardprotecting groups.

Oligomers of the invention can be covalently derivatized to moietiesthat facilitate cell association using cleavable linkers. Linkers usedfor such conjugates can include disulfide linkages that are reducedafter the oligomer-transport agent conjugate has entered a cell.Disulfide-containing linkers of this type have a controllable half life.Such linkers are stable under extracellular conditions relative tointracellular conditions due to the redox potential of the disulfidelinkage.

Reactive Functional Groups

The components of the compounds of the invention (e.g., linkers,nucleoside, nucleotide, oligonucleotide, nucleic acid, carrier molecule,and solid support) may be linked through linkage sites formed byreaction of a first and a second reactive functional group. The reactivefunctional groups are of complementary reactivity, and they react toform a covalent link between two components of the compounds, referredto herein as a linkage site. For example, compounds according to Formula(I) or (II) wherein R^(x) or R^(s) is a reactive functional group can bereacted with a reactive functional group of complementary reactivity onanother component (such as a linker, nucleoside, nucleotide,oligonucleotide, nucleic acid, carrier molecule, and solid support) tocovalently join the components through the resulting linkage site. Thereactive functional group of complementary reactivity can be located atany position of the other component (linker, nucleoside etc.), e.g., analkyl or heteroalkyl an aryl or heteroaryl nucleus or a substituent onan aryl or heteroaryl nucleus. In various embodiments, when the reactivegroup is attached to an alkyl (or heteroalkyl), or substituted alkyl (orheteroalkyl) chain, the reactive group is preferably located at aterminal position of the chain.

Reactive groups and classes of reactions useful in practicing thepresent invention are generally those that are well known in the art ofbioconjugate chemistry. Currently favored classes of reactions availablewith reactive precursors of the oligomers of the invention are thosewhich proceed under relatively mild conditions. These include, but arenot limited to nucleophilic substitutions (e.g., reactions of amines andalcohols with acyl halides, active esters), electrophilic substitutions(e.g., enamine reactions) and additions to carbon-carbon andcarbon-heteroatom multiple bonds (e.g., Michael reaction, Diels-Alderaddition). These and other useful reactions are discussed in, forexample, March, ADVANCED ORGANIC CHEMISTRY, 3rd Ed., John Wiley & Sons,New York, 1985; Hermanson, BIOCONJUGATE TECHNIQUES, Academic Press, SanDiego, 1996; and Feeney et al., MODIFICATION OF PROTEINS; Advances inChemistry Series, Vol. 198, American Chemical Society, Washington, D.C.,1982.

By way of example, reactive functional groups of use in the presentinvention include, but are not limited to olefins, acetylenes, alcohols,phenols, ethers, oxides, halides, aldehydes, ketones, carboxylic acids,esters, amides, cyanates, isocyanates, thiocyanates, isothiocyanates,amines, hydrazines, hydrazones, hydrazides, diazo, diazonium, nitro,nitriles, mercaptans, sulfides, disulfides, sulfoxides, sulfones,sulfonic acids, sulfinic acids, acetals, ketals, anhydrides, sulfates,sulfenic acids isonitriles, amidines, imides, imidates, nitrones,hydroxylamines, oximes, hydroxamic acids thiohydroxamic acids, allenes,ortho esters, sulfites, enamines, ynamines, ureas, pseudoureas,semicarbazides, carbodiimides, carbamates, imines, azides, azocompounds, azoxy compounds, and nitroso compounds. Reactive functionalgroups also include those used to prepare bioconjugates, e.g.,N-hydroxysuccinimide esters, maleimides and the like. Methods to prepareeach of these functional groups are well known in the art and theirapplication to or modification for a particular purpose is within theability of one of skill in the art (see, for example, Sandler and Karo,eds. ORGANIC FUNCTIONAL GROUP PREPARATIONS, Academic Press, San Diego,1989).

Useful reactive functional group conversions include, for example:

-   -   (a) carboxyl groups which are readily converted to various        derivatives including, but not limited to, active esters (e.g.,        N-hydroxysuccinimide esters, N-hydroxybenztriazole esters,        thioesters, p-nitrophenyl esters), acid halides, acyl        imidazoles, alkyl, alkenyl, alkynyl and aromatic esters;    -   (b) hydroxyl groups, which can be converted to esters, ethers,        halides, aldehydes, etc.    -   (c) haloalkyl groups, wherein the halide can be later displaced        with a nucleophilic group such as, for example, an amine, a        carboxylate anion, thiol anion, carbanion, or an alkoxide ion,        thereby resulting in the covalent attachment of a new group at        the site of the halogen atom;    -   (d) dienophile groups, which are capable of participating in        Diels-Alder reactions such as, for example, maleimido groups;    -   (e) aldehyde or ketone groups, such that subsequent        derivatization is possible via formation of carbonyl derivatives        such as, for example, imines, hydrazones, semicarbazones or        oximes, or via such mechanisms as Grignard addition or        alkyllithium addition;    -   (f) sulfonyl halide groups for subsequent reaction with amines,        for example, to form sulfonamides;    -   (g) thiol groups, which can be, for example, converted to        disulfides or reacted with acyl halides;    -   (h) amine or sulfhydryl groups, which can be, for example,        acylated, alkylated or oxidized;    -   (i) alkenes, which can undergo, for example, cycloadditions,        acylation, Michael addition, etc;    -   (j) epoxides, which can react with, for example, amines and        hydroxyl compounds; and    -   (k) phosphoramidites and other standard functional groups useful        in nucleic acid synthesis.

The reactive functional groups can be chosen such that they do notparticipate in, or interfere with, the reactions necessary to assemblethe oligomer of the invention. Alternatively, a reactive functionalgroup can be protected from participating in the reaction by thepresence of a protecting group. Those of skill in the art understand howto protect a particular functional group such that it does not interferewith a chosen set of reaction conditions. For examples of usefulprotecting groups, see, for example, Greene et al., PROTECTIVE GROUPS INORGANIC SYNTHESIS, John Wiley & Sons, New York, 1991.

Covalent Bonding Moiety

Included in some of the oligomers of the invention is a reactivefunctional group moiety which is capable of effecting at least onecovalent bond between the oligomer and a target sequence. Multiplecovalent bonds can also be formed by providing a multiplicity of suchmoieties. The covalent bond is preferably to a nucleobase residue in thetarget strand, but can also be made with other portions of the target,including the sugar or phosphodiester. The reaction nature of the moietywhich effects crosslinker determines the nature of the target in theduplex. Preferred crosslinker moieties include acylating and alkylatingagents, and, in particular, those positioned relative to the sequencespecificity-conferring portion so as to permit reaction with the targetlocation in the strand.

The crosslinker moiety can conveniently be placed as an analogouspyrimidine or purine residue in the sequence of the oligomer. Theplacement can be at the 5′- and/or 3′-ends, the internal portions of thesequence, or combinations of the above. Placement at the termini topermit enhanced flexibility is preferred. Analogous moieties can also beattached to peptide backbones.

Exemplary of alkylating moieties that are useful in the inventioninclude N⁴,N⁴-ethanocytosine and N⁶,N⁶-ethanoadenine.

It is clear that the nucleobase need not be a purine or pyrimidine;indeed the moiety to which the reactive function is attached need not bea nucleobase at all and may be a sugar, a linker, a quencher, astabilizing moiety a fluorophore or some combination of these componentsof the oligomers of the invention. Any means of attaching the reactivegroup is satisfactory so long as the positioning is correct.

Synthesis

Compounds of the invention (such as solid supports, monomers (e.g.,phosphoramidites) and oligomers of the invention or the segmentsthereof) are generally conventionally synthesized. See, for example,U.S. Pat. Nos. 7,019,129; 8,466,266; and 7,879,986. The syntheticmethods known in the art and described herein can be used to synthesizeoligomers containing compounds of the invention, as well as othernucleobases known in the art, using appropriately protectednucleomonomers. Methods for the synthesis of oligomers are found, forexample, in Froehler, B., et al., Nucleic Acids Res. (1986)14:5399-5467; Nucleic Acids Res. (1988) 16:4831-4839; Nucleosides andNucleotides (1987) 6:287-291; Froehler, B., Tetrahedron Letters (1986)27:5575-5578; Caruthers, M. H. in Oligodeoxynucleotides-AntisenseInhibitions of Gene Expression (1989), J. S. Cohen, editor, CRC Press,Boca Raton, p7-24; Reese, C. B. et al., Tetrahedron Letters (1985)28:2245-2248. Synthesis of the methylphosphonate linked oligomers viamethyl phosphonamidite chemistry has also been described (Agrawal, S. etal., Tetrahedron Letters (1987) 28:3539-3542; Klem, R. E., et al.,International Publication Number WO 92/07864).

In an exemplary embodiment, nucleomonomers are directly incorporatedinto oligomers or a convenient fragment thereof using standard synthesisconditions and reagents. Exemplary linkages made by this method includephosphodiester, phosphorothioate, phosphoroamidate, methylphosphonate,phosphorodithioate, carbonate, morpholino carbamate and sulfonate.

In various embodiments, synthesis involves synthesis of short synthons(dimers, trimers, etc.) starting with an appropriate precursor. Thisapproach is suitable for synthesis of linkages includingN-methylhydroxylamine, dimethylhydrazo, sulfamate, carbamate, sulfonate,sulfonamide, formacetal thioformacetal and carbonate.

Oligomers of the invention can be synthesized by any suitable chemistryincluding amidite, triester or hydrogen phosphonate coupling methods andconditions. The oligomers are preferably synthesized from appropriatestarting synthons which are preferably protected at the 5′-position withDMT, MMT, FMOC (9-fluorenylmethoxycarbonyl), PACO (phenoxyacetyl), asilyl ether such as TBDMS (t-butyldiphenylsilyl) or TMS (trimethylsilyl)and activated at the 3′-position is an ester, H-phosphonate, an amiditesuch as β-cyanoethylphosphoramidite, a silyl ether such as TBDMS or TMSor t-butyldiphenyl. Alternatively, appropriate uridine or cytidineprecursors such as blocked 5-iodo-2′-deoxyuridine,5-iodo-2′-O-alkyluridine, 5-bromo-2′-deoxyuridine,5-trifluoromethanesulfonate-2′-deoxyuridine, 5-bromo-2′-O-alkyluridineor blocked and protected 5-iodo-2′-deoxycytidine,5-bromo-2′-deoxycytidine, 5-trifluoromethanesulfonate-2′-deoxycytidine,5-iodo-2′-O-alkylcytidine, 5-bromo-2′-O-alkylcytidine can beconveniently incorporated into short oligomers such as dimer, trimer,tetramer, pentamer or longer synthons that are subsequently derivatizedto yield suitable synthons and longer oligomers.

Exemplary synthesis of oligomers containing about 4 or morenucleomonomer residues are accomplished using synthons such as monomers,dimers or trimers that carry a coupling group suitable for use withamidite, H-phosphonate or triester chemistries. The synthon can be usedto link the components of the oligomer via a phosphodiester orphosphorous-containing linkage other than phosphodiester (e.g.,phosphorothioate, methylphosphonate, thionomethylphosphonate,phosphoramidate and the like).

Synthesis of other nonphosphorous-containing substituted linkages can beaccomplished using appropriate precursors as known in the art.

Once the desired nucleic acid is synthesized, it is preferably cleavedfrom the solid support on which it was synthesized and treated, bymethods known in the art, to remove any protecting groups present (e.g.,60° C., 5 h, concentrated ammonia). In those embodiments in which abase-sensitive group is attached to the nucleic acids (e.g., TAMRA), thedeprotection will preferably use milder conditions (e.g., butylamine:water 1:3, 8 hours, 70° C.). Deprotection under these conditions isfacilitated by the use of quick deprotect amidites (e.g., dC-acetyl,dG-dmf).

Following cleavage from the support and deprotection, the nucleic acidis purified by any method known in the art, including chromatography,extraction and gel purification. In a preferred embodiment, the nucleicacid is purified using HPLC. The concentration and purity of theisolated nucleic acid is preferably determined by measuring the opticaldensity at 260 nm in a spectrophotometer.

Assays and Oligomeric Probes of the Invention

In various embodiments, the present invention provides an oligomer ofuse in one or more assay formats. In selected embodiments the oligomerparticipates in the generation of a detectable signal upon associationwith or dissociation from its target. The oligomeric probes of theinvention are not limited in use to any particular assay format.Accordingly, the following description is intended to illustrateexemplary assays formats in which the oligomers of the invention finduse, and is not intended to be limiting of the assay formats in whichthe oligomers are of use.

Assays

The following discussion is generally relevant to the assays describedherein. This discussion is intended to illustrate the invention byreference to certain preferred embodiments and should not be interpretedas limiting the scope of probes and assay types in which the compoundsof the invention find use. Other assay formats utilizing the compoundsof the invention will be apparent to those of skill in the art.

In general, to determine the concentration of a target molecule, suchas, for example, a nucleic acid of unknown quantity, it is preferable tofirst obtain reference data in which constant amounts of probe arecontacted with nucleic acid standards spanning a range of knownquantities. The intensity of fluorescence emission from each of thereference mixtures is used to derive a graph or standard curve, in whichthe unknown concentration is compared to the intensity of the knownstandards. For example, a probe that: a) hybridizes to a sequence withinthe target nucleic acid; b) has fluorophore and quencher modificationsupon the 5′ and 3′ termini being the sites of labeling; and c) hasfluorogenic character that is quenched in an unbound conformation andthen releases signal upon binding to the target nucleic acid, can beused to obtain such reference data. Such a probe gives a characteristicfluorescence emission in which the signal increases as the concentrationof target nucleic acid increases. Then, a sample with an unknownquantity of target is contacted with the probe, and the fluorescenceintensity from the mixture is determined. The intensity of fluorescenceemission is then compared with the reference standards to obtain theconcentration of the target in the test mixture.

Multiplex Analyses

In another embodiment, the solid supports and oligomers of the inventionare utilized as a probe or a component of one or more probes used in amultiplex assay for detecting one or more species in a mixture.

Probes based on the solid supports or oligomers of the invention areparticularly useful in performing multiplex-type analyses and assays. Ina typical multiplex analysis, two or more distinct species (or regionsof one or more species) are detected using two or more probes, whereineach of the probes is labeled with a different fluorophore. Preferredspecies used in multiplex analyses relying on donor-acceptor energytransfer meet at least two criteria: the fluorescent species is brightand spectrally well-resolved; and the energy transfer between thefluorescent species and the quencher is efficient.

The solid supports and oligomers of the invention allow for the designof multiplexed assays in which more than one fluorescent reporter ispartnered with one or more quencher structures. A number of differentmultiplexed assays using the solid supports or oligomers of theinvention will be apparent to one of skill in the art. In one exemplaryassay, each of at least two distinct fluorescent reporters are pairedwith the same type of quencher structure on their respective oligomers,to modulate the signal through either FRET or contact quenching.Alternatively, an assay can be practiced in which at least two distinctfluorescent reporters are partnered with distinct quencher structures,to which the fluorescent properties are better “matched.” Thefluorophores can be bound to the same molecule as the quencher or to adifferent molecule. Moreover, similar to the quencher and thefluorophores, the carrier molecules of use in a particular assay system,such as the oligo sequence that covalently tethers the fluorophore andquencher, can either be the same or different.

In addition to the mixtures described above, the present invention alsoprovides a qualitative method for detecting the presence a particularmolecular species. The method includes: (a) contacting the species witha mixture containing a solid support or oligomer of the invention; and(b) detecting a change in a fluorescent property of one or morecomponent of the resulting mixture, thereby detecting the presence themolecular species.

The simultaneous use of two or more probes using donor-acceptor energytransfer is known in the art. For example, multiplex assays usingnucleic acid probes with different sequence specificities have beendescribed. Fluorescent probes have been used to determine whether anindividual is homozygous wild-type, homozygous mutant or heterozygousfor a particular mutation. For example, using one quenched-fluoresceinmolecular beacon that recognizes the wild-type sequence and anotherrhodamine-quenched molecular beacon that recognizes a mutant allele, itis possible to genotype individuals for the β-chemokine receptor(Kostrikis et al. Science 279:1228-1229 (1998)). The presence of only afluorescein signal indicates that the individual is wild-type, and thepresence of rhodamine signal only indicates that the individual is ahomozygous mutant. The presence of both rhodamine and fluorescein signalis diagnostic of a heterozygote. Tyagi et al. Nature Biotechnology 16:49-53 (1998)) have described the simultaneous use of four differentlylabeled molecular beacons for allele discrimination, and Lee et al.,BioTechniques 27: 342-349 (1999) have described seven color homogenousdetection of six PCR products.

The quenchers of the present invention can be used in multiplex assaysdesigned to detect and/or quantify substantially any species, including,for example, whole cells, viruses, proteins (e.g., enzymes, antibodies,receptors), glycoproteins, lipoproteins, subcellular particles,organisms (e.g., Salmonella), nucleic acids (e.g., DNA, RNA, andanalogues thereof), polysaccharides, lipopolysaccharides, lipids, fattyacids, non-biological polymers and small molecules (e.g., toxins, drugs,pesticides, metabolites, hormones, alkaloids, steroids).

Nucleic Acid Probes

The solid supports and oligomers of the invention are usefulnucleic-acid probes and they can be used as components of detectionagents in a variety of DNA amplification/quantification strategiesincluding, for example, 5′-nuclease assay, Strand DisplacementAmplification (SDA), Nucleic Acid Sequence-Based Amplification (NASBA),Rolling Circle Amplification (RCA), as well as for direct detection oftargets in solution phase or solid phase (e.g., array) assays.Furthermore, the solid supports and oligomers can be used in probes ofsubstantially any format, including, for example, format selected frommolecular beacons, Scorpion Probes™, Sunrise Probes™, conformationallyassisted probes, light up probes, Invader Detection probes, and TaqMan™probes. See, for example, Cardullo, R., et al., Proc. Natl. Acad. Sci.USA, 85:8790-8794 (1988); Dexter, D. L., J. Chem. Physics, 21:836-850(1953); Hochstrasser, R. A., et al., Biophysical Chemistry, 45:133-141(1992); Selvin, P., Methods in Enzymology, 246:300-334 (1995);Steinberg, I., Ann. Rev. Biochem., 40:83-114 (1971); Stryer, L., Ann.Rev. Biochem., 47:819-846 (1978); Wang, G., et al., Tetrahedron Letters,31:6493-6496 (1990); Wang, Y., et al., Anal. Chem., 67:1197-1203 (1995);Debouck, C., et al., in supplement to nature genetics, 21:48-50 (1999);Rehman, F. N., et al., Nucleic Acids Research, 27:649-655 (1999);Cooper, J. P., et al., Biochemistry, 29:9261-9268 (1990); Gibson, E. M.,et al., Genome Methods, 6:995-1001 (1996); Hochstrasser, R. A., et al.,Biophysical Chemistry, 45:133-141 (1992); Holland, P. M., et al., ProcNatl. Acad. Sci USA, 88:7276-7289 (1991); Lee, L. G., et al., NucleicAcids Rsch., 21:3761-3766 (1993); Livak, K. J., et al., PCR Methods andApplications, Cold Spring Harbor Press (1995); Vamosi, G., et al.,Biophysical Journal, 71:972-994 (1996); Wittwer, C. T., et al.,Biotechniques, 22:176-181 (1997); Wittwer, C. T., et al., Biotechniques,22:130-38 (1997); Giesendorf, B. A. J., et al., Clinical Chemistry,44:482-486 (1998); Kostrikis, L. G., et al., Science, 279:1228-1229(1998); Matsuo, T., Biochemica et Biophysica Acta, 1379:178-184 (1998);Piatek, A. S., et al., Nature Biotechnology, 16:359-363 (1998);Schofield, P., et al., Appl. Environ. Microbiology, 63:1143-1147 (1997);Tyagi S., et al., Nature Biotechnology, 16:49-53 (1998); Tyagi, S., etal., Nature Biotechnology, 14:303-308 (1996); Nazarenko, I. A., et al.,Nucleic Acids Research, 25:2516-2521 (1997); Uehara, H., et al.,Biotechniques, 26:552-558 (1999); D. Whitcombe, et al., NatureBiotechnology, 17:804-807 (1999); Lyamichev, V., et al., NatureBiotechnology, 17:292 (1999); Daubendiek, et al., Nature Biotechnology,15:273-277 (1997); Lizardi, P. M., et al., Nature Genetics, 19:225-232(1998); Walker, G., et al., Nucleic Acids Res., 20:1691-1696 (1992);Walker, G. T., et al., Clinical Chemistry, 42:9-13 (1996); and Compton,J., Nature, 350:91-92 (1991).

Thus, the present invention provides a method for detecting a nucleicacid target sequence. The method includes: (a) contacting the targetsequence with a detector nucleic acid (e.g., an oligomer of theinvention); (b) hybridizing the target binding sequence to the targetsequence, thereby altering the conformation of the detector nucleicacid, causing a change in a fluorescence parameter; and (c) detectingthe change in the fluorescence parameter, thereby detecting the nucleicacid target sequence.

In the methods described herein, unless otherwise noted, a preferreddetector nucleic acid includes a single-stranded target bindingsequence. The binding sequence has linked thereto: i) a fluorophore, ii)a first quencher, and iii) a second quencher. The binding sequence hasoptionally further linked thereto a stabilizing moiety. Moreover, priorto its hybridization to a complementary sequence, the detector nucleicacid is preferably in a conformation that allows donor-acceptor energytransfer between the fluorophore and the quencher when the fluorophoreis excited. Furthermore, in each of the methods described in thissection, a change in fluorescence is detected as an indication of thepresence of the target sequence. The change in fluorescence ispreferably detected in real-time.

Presently preferred nucleic acid probes do not require the nucleic acidto adopt a secondary structure for the probe to function. In thismethod, and unless otherwise noted, the other methods described in thissection, the detector nucleic acid can assume substantially anyintramolecularly associated secondary structure, but this structure ispreferably a member selected from hairpins, stem-loop structures,pseudoknots, triple helices and conformationally assisted structures.Moreover, the intramolecularly base-paired secondary structurepreferably comprises a portion of the target binding sequence.

In another aspect, the invention provides a method for detectingamplification of a target sequence. The method involves the use of anamplification reaction such as PCR. An exemplary amplification reactionincludes one or more of the following steps:

-   (a) hybridizing a sample nucleic acid comprising the target sequence    of interest with PCR primers that flank the target sequence;-   (b) extending the hybridized primers with a polymerase to produce    the PCR product, and separating the two strands of the PCR product    to make accessible the sense and antisense strands of the target    sequence;-   (c) hybridizing a detector nucleic acid to the sense or antisense    strand of the target sequence in the PCR product, wherein the    detector nucleic acid includes:    -   i) a single stranded target binding sequence that is        complementary to at least a portion of the sense or antisense        strand of the target sequence in the PCR product, and hybridizes        to a region between the PCR primers;    -   ii) a fluorophore; and    -   iii) a first quencher and a second quencher;    -   wherein prior to its hybridization to the target sequence, the        detector nucleic acid is in a conformation allowing        donor-acceptor energy transfer between the fluorophore and the        quencher when the fluorophore is excited;    -   thereby altering the conformation of the detector nucleic acid        (for example, linearizing any secondary structure or random coil        conformations that contribute to the quenching efficiency),        causing a change in a fluorescence parameter (such as the signal        intensity); and-   (d) measuring the change in the fluorescence parameter to detect the    target sequence and its amplification.    Optionally, the change in the fluorescence parameter can be made    permanent if the polymerase encounters the hybridized detector    nucleic acid during primer extension (step (b) above) and hydrolyzes    the oligomer tethering the fluorophore and quencher, such as through    a secondary nuclease activity of the polymerase.

In yet a further aspect, the invention provides a method of ascertainingwhether a first nucleic acid and a second nucleic acid hybridize. Inthis method, the first nucleic acid is an oligomer (in solution orattached to a solid support) according to the invention. The methodincludes: (a) contacting the first nucleic acid with the second nucleicacid; (b) detecting an alteration in a fluorescent property of a memberselected from the first nucleic acid, the second nucleic acid and acombination thereof, thereby ascertaining whether the hybridizationoccurs.

In various embodiments, the present invention provides probes andmethods of use in detecting polymorphism in nucleic acid targetsequences. Polymorphism refers to the occurrence of two or moregenetically determined alternative sequences or alleles in a population.A polymorphic marker or site is the locus at which divergence occurs.Preferred markers have at least two alleles, each occurring at frequencyof greater than 1%, and more preferably greater than 10% or 20% of aselected population. A polymorphic locus may be as small as one basepair. Polymorphic markers include restriction fragment lengthpolymorphisms, variable number of tandem repeats (VNTR's), hypervariableregions, minisatellites, dinucleotide repeats, trinucleotide repeats,tetranucleotide repeats, simple sequence repeats, and insertion elementssuch as Alu. The first identified allelic form is arbitrarily designatedas the reference form and other allelic forms are designated asalternative or variant alleles. The allelic form occurring mostfrequently in a selected population is sometimes referred to as thewildtype form. Diploid organisms may be homozygous or heterozygous forallelic forms. A diallelic polymorphism has two forms. A triallelicpolymorphism has three forms.

In an exemplary embodiment, a probe of the invention is utilized todetect a single nucleotide polymorphism. A single nucleotidepolymorphism occurs at a polymorphic site occupied by a singlenucleotide, which is the site of variation between allelic sequences.The site is usually preceded by and followed by highly conservedsequences of the allele (e.g., sequences that vary in less than 1/100 or1/1000 members of the populations). A single nucleotide polymorphismusually arises due to substitution of one nucleotide for another at thepolymorphic site. A transition is the replacement of one purine byanother purine or one pyrimidine by another pyrimidine. A transversionis the replacement of a purine by a pyrimidine or vice versa. Singlenucleotide polymorphisms can also arise from a deletion of a nucleotideor an insertion of a nucleotide relative to a reference allele.

An oligomer of the invention bearing two quenchers and a fluorophore canbe used or, alternatively, one or more of the nucleic acids can belabeled with members of an energy transfer pair (e.g., a quencher orfluorophore). When a nucleic acid labeled with quenchers is the probe,the interaction between the first and second nucleic acids can bedetected by observing the interaction between the quenchers and thenucleic acid or, more preferably, the quenching by the quenchers of thefluorescence of a fluorophore attached to the second nucleic acid.

In some embodiments, a ground state complex between the quenchers and afluorophore is formed. In an exemplary embodiment, both the quenchersand fluorophore are conjugated to the same nucleic acid oligomer.

In addition to their general utility in probes designed to investigatenucleic acid amplification, polymorphism and detection andquantification, the present solid supports and oligomers can be used insubstantially any nucleic acid probe format now known or laterdiscovered. For example, the solid supports and oligomers of theinvention can be incorporated into probe motifs, such as Taqman™ probes(Held et al., Genome Res. 6: 986-994 (1996), Holland et al., Proc. Nat.Acad. Sci. USA 88: 7276-7280 (1991), Lee et al., Nucleic Acids Res. 21:3761-3766 (1993)), molecular beacons (Tyagi et al., Nature Biotechnology14:303-308 (1996), Jayasena et al., U.S. Pat. No. 5,989,823, issued Nov.23, 1999)) scorpion probes (Whitcomb et al., Nature Biotechnology 17:804-807 (1999)), sunrise probes (Nazarenko et al., Nucleic Acids Res.25: 2516-2521 (1997)), conformationally assisted probes (Cook, R., U.S.Patent Application Publication No. 2007/0059752, published Mar. 15,2007), peptide nucleic acid (PNA)-based light up probes (Kubista et al.,WO 97/45539, December 1997), double-strand specific DNA dyes (Higuchi etal., Bio/Technology 10: 413-417 (1992), Wittwer et al., BioTechniques22: 130-138 (1997)) and the like. These and other probe motifs withwhich the present quenchers can be used are reviewed in NONISOTOPIC DNAPROBE TECHNIQUES, Academic Press, Inc. 1992.

The oligomers for use in the probes of the invention can be any suitablesize, and are preferably in the range of from about 2 to about 100nucleotides, more preferably from about 10 to about 80 nucleotides andmore preferably still, from about 10 to about 40 nucleotides. In thedual labeled (fluorophore-quencher) probes, the donor moiety ispreferably separated from the quencher by at least about 6, preferablyat least about 8, preferably at least about 10 nucleotides, and morepreferably by at least about 15 nucleotides. In various embodimentsdonor moiety is preferably attached to either the 3′- or 5′-terminalnucleotides of the probe. The quencher moiety is also preferablyattached to either the 3′- or 5′-terminal nucleotides of the probe. Morepreferably, the donor and acceptor moieties are attached to the 3′- and5′- or 5′- and 3′-terminal nucleotides of the probe, respectively,although internal placement is also useful.

The precise sequence and length of a nucleic acid probe of the inventiondepends in part on the nature of the target polynucleotide to which itbinds. The binding location and length may be varied to achieveappropriate annealing and melting properties for a particularembodiment. Guidance for making such design choices can be found in manyart-recognized references.

In some embodiments, the 3′-terminal nucleotide of the nucleic acidprobe is blocked or rendered incapable of extension by a nucleic acidpolymerase. Such blocking is conveniently carried out by the attachmentof a donor or acceptor moiety to the terminal 3′-position of the nucleicacid probe, either directly or by a linker moiety.

The nucleic acid can comprise DNA, RNA or chimeric mixtures orderivatives or modified versions thereof. Both the probe and targetnucleic acid can be present as a single strand, duplex, triplex, etc.Moreover, the nucleic acid can be modified at the nucleobase moiety,sugar moiety, or phosphate backbone with other groups such asradioactive labels, minor groove binders, intercalating agents,acetylinically unsaturated hydrocarbons, fluoralkyl groups, donor and/oracceptor moieties and the like.

The oligomers of the invention are useful as primers that are discretesequences or as primers with a random sequence. Random sequence primersare generally about 6 or 7 nucleomonomers in length. Such primers can beused in various nucleic acid amplification protocols (PCR, ligase chainreaction, etc) or in cloning protocols. Substitutions on the 5′ end ofthe invention generally do not interfere with the capacity of theoligomer to function as a primer. Oligomers of the invention having2¹-modifications at sites other than the 3′ terminal residue, othermodifications that render the oligomer RNase H incompetent or otherwisenuclease stable can be advantageously used as probes or primers for RNAor DNA sequences in cellular extracts or other solutions that containnucleases. Thus, the oligomers can be used in protocols for amplifyingnucleic acid in a sample by mixing the oligomer with a sample containingtarget nucleic acid, followed by hybridization of the oligomer with thetarget nucleic acid and amplifying the target nucleic acid by PCR, LCRor other suitable methods.

The oligomers derivatized with chelating agents such as EDTA, DTPA oranalogs of 1,2-diaminocyclohexane acetic acid can be utilized in variousin vitro diagnostic assays as described (U.S. Pat. Nos. 4,772,548,4,707,440 and 4,707,352). Alternatively, oligomers of the invention canbe derivatized with crosslinker agents such as5-(3-iodoacetamidoprop-1-yl)-2′-deoxyuridine or5-(3-(4-bromobutyramido)prop-1-yl)-2¹-deoxyuridine and used in variousassay methods or kits as described (International Publication No. WO90/14353).

In addition to the foregoing uses, the ability of the oligomers toinhibit gene expression can be verified in in vitro systems by measuringthe levels of expression in subject cells or in recombinant systems, byany suitable method (Graessmann, M., et al., Nucleic Acids Res. (1991)19:53-59).

Conditions that favor hybridization between oligomer of the presentinvention and target nucleic acid molecules can be determinedempirically by those skilled in the art, and can include optimalincubation temperatures, salt concentrations, length and nucleobasecompositions of oligonucleotide analogue probes, and concentrations ofoligomer and nucleic acid molecules of the sample. Preferably,hybridization is performed in the presence of at least one millimolarmagnesium and at a pH that is above 6.0. In some embodiments, it may benecessary or desirable to treat a sample to render nucleic acidmolecules in the sample single-stranded prior to hybridization. Examplesof such treatments include, but are not limited to, treatment with base(preferably followed by neutralization), incubation at high temperature,or treatment with nucleases.

In addition, because the salt dependence of hybridization to nucleicacids is largely determined by the charge density of the backbone of ahybridizing oligonucleotide analogue, incorporating nonstandardnucleotide analogs into the oligomer of the present invention canincrease or decrease the salt dependence of hybridization. Thismodulation can be used to advantage in the methods of the presentinvention where it can in some aspects be desirable to be able toincrease the stringency of hybridization by changing salt conditions,for example, or release a hybridized nucleic acid by reducing the saltconcentration. In yet other aspects of the present invention, it can bedesirable to have high-affinity binding of an oligonucleotide analogueof the present invention to a nucleic acid in very low salt. In thiscase, positioning nucleotide monomers with uncharged backbone moietiesinto an oligonucleotide of the present invention is advantageous.

The high degree of specificity of oligomers of the present invention inbinding to target nucleic acid molecules allow the practitioner toselect hybridization conditions that can favor discrimination betweennucleic acid sequences that comprise a stretch of sequence that iscompletely complementary to at least a portion of one or more oligomerand target nucleic acid molecules that comprise a stretch of sequencethat comprises a small number of non-complementary nucleobases within asubstantially complementary sequence. For example, hybridization or washtemperatures can be selected that permit stable hybrids between oligomerof the present invention and target nucleic acid molecules that arecompletely complementary along a stretch of sequence but promotedissociation of hybrids between oligomer of the present invention andtarget nucleic acid molecules that are not completely complementary,including those that comprise one or two nucleobase mismatches along astretch of complementary sequence. The selection of a temperature forhybridization and washes can be dependent, at least in part, on otherconditions, such as the salt concentration, the concentration ofoligomer and target nucleic acid molecules, the relative proportions ofoligomer to target nucleic acid molecules, the length of the oligomersto be hybridized, the nucleobase composition of the oligomer and targetnucleic acid molecules, the monomer composition of the oligonucleotideanalogue molecules, etc. In addition, when selecting for conditions thatfavor stable hybrids of completely complementary molecules and disfavorstable hybrids between oligomer and target nucleic acid molecules thatare mismatched by one or more nucleobases, additional conditions can betaken into account, and, where desirable, altered, including but notlimited to, the length of the oligonucleotide analogue to be hybridized,the length of the stretch of sequence of complementarity betweenoligomer and target nucleic acid molecules, the number ofnon-complementary nucleobases within a stretch of sequence ofcomplementarity, the identity of mismatched nucleobases, the identity ofnucleobases in the vicinity of the mismatched nucleobases, and therelative position of any mismatched nucleobases along a stretch ofcomplementarity. Those skilled in the art of nucleic acid hybridizationwould be able to determine favorable hybridization and wash conditionsin using oligomer of the present invention for hybridization to targetnucleic acid molecules, depending on the particular application.“Favorable conditions” can be those favoring stable hybrids betweenoligomer and target nucleic acid molecules that are, at least in part,substantially complementary, including those that comprise one or moremismatches.

“Favorable conditions” can be those favoring stable hybrids betweenoligomer and target nucleic acid molecules that are, at least in part,completely complementary and disfavor or destabilized hybrids betweenmolecules that are not completely complementary.

Using methods such as those disclosed herein, the melting temperature ofoligomer of the present invention hybridized to target nucleic acidmolecules of different sequences can be determined and can be used indetermining favorable conditions for a given application. It is alsopossible to empirically determine favorable hybridization conditions by,for example, hybridizing target nucleic acid molecules to oligomer thatare attached to a solid support and detecting hybridized complexes.

Target nucleic acid molecules that are bound to solid supports oroligomeric probes of the present invention can be conveniently andefficiently separated from unbound nucleic acid molecules of the surveypopulation by the direct or indirect attachment of oligomer probes to asolid support. A solid support can be washed at high stringency toremove nucleic acid molecules that are not bound to oligomer probes.However, the attachment of oligomer probes to a solid support is not arequirement of the present invention. For example, in some applicationsbound and unbound nucleic acid molecules can be separated bycentrifugation through a matrix or by phase separation or some by otherforms of separation (for example, differential precipitation) that canoptionally be aided by chemical groups incorporated into the oligomerprobes (see, for example, U.S. Pat. No. 6,060,242 issued May 9, 2000, toNie et

Nucleic Acid Capture Probes

In one embodiment, an immobilized nucleic acid comprising a firstquencher and a second quencher is used as a capture probe. Theimmobilized nucleic acid optionally further comprises a stabilizingmoiety. The nucleic acid probe can be attached directly to a solidsupport, for example by attachment of the 3′- or 5′-terminal nucleotideof the probe to the solid support. More preferably, however, the probeis attached to the solid support by a linker (supra). The linker servesto distance the probe from the solid support. The linker is mostpreferably from about 5 to about 30 atoms in length, more preferablyfrom about 10 to about 50 atoms in length.

In various embodiments, the solid support is also used as the synthesissupport in preparing the oligomer (probe). The length and chemicalstability of the linker between the solid support and the first 3′-unitof nucleic acid play an important role in efficient synthesis andhybridization of support bound nucleic acids. The linker arm ispreferably sufficiently long so that a high yield (>97%) can be achievedduring automated synthesis. The required length of the linker willdepend on the particular solid support used. For example, a six atomlinker is generally sufficient to achieve a >97% yield during automatedsynthesis of nucleic acids when high cross-linked polystyrene is used asthe solid support. The linker arm is preferably at least 20 atoms longin order to attain a high yield (>97%) during automated synthesis whenCPG is used as the solid support.

Hybridization of a probe immobilized on a solid support generallyrequires that the probe be separated from the solid support by at least30 atoms, more preferably at least 50 atoms. In order to achieve thisseparation, the linker generally includes a spacer positioned betweenthe linker and the 3′-terminus. For nucleic acid synthesis, the linkerarm is usually attached to the 3′-OH of the 3′-terminus by an esterlinkage which can be cleaved with basic reagents to free the nucleicacid from the solid support.

A wide variety of linkers are known in the art, which may be used toattach the nucleic acid probe to the solid support. The linker may beformed of any compound, which does not significantly interfere with thehybridization of the target sequence to the probe attached to the solidsupport. The linker may be formed of, for example, a homopolymericnucleic acid, which can be readily added on to the linker by automatedsynthesis. Alternatively, polymers such as functionalized polyethyleneglycol can be used as the linker. Such polymers are presently preferredover homopolymeric nucleic acids because they do not significantlyinterfere with the hybridization of probe to the target nucleic acid.Polyethylene glycol is particularly preferred because it is commerciallyavailable, soluble in both organic and aqueous media, easy tofunctionalize, and completely stable under nucleic acid synthesis andpost-synthesis conditions.

The linkage sites between the solid support, the linker and the probeare preferably not cleaved during synthesis or removal of nucleobaseprotecting groups under basic conditions at high temperature. Theselinkages can, however, be selected from groups that are cleavable undera variety of conditions. Examples of presently preferred linkagesinclude carbamate, ester and amide linkages.

Detection of Nucleic Acids in Samples

Solid supports and oligomers of the present invention can be used fordetection of nucleic acids. Such detection methods include: providing asample, contacting at least one oligonucleotide analogue of the presentinvention with the sample under conditions that allow hybridization ofoligomer to nucleic acid molecules, and detecting one or more nucleicacid molecules of the sample that have hybridized to one or moreoligomer of the present invention.

A sample can be from any source, and can be a biological sample, such asa sample from an organism or a group of organisms from the same ordifferent species. A biological sample can be a sample of bodily fluid,for example, a blood sample, serum sample, lymph sample, a bone marrowsample, ascites fluid, pleural fluid, pelvic wash fluid, ocular fluid,urine, semen, sputum, or saliva. A biological sample can also be anextract from cutaneous, nasal, throat, or genital swabs, or extracts offecal material. Biological samples can also be samples of organs ortissues, including tumors. Biological samples can also be samples ofcell cultures, including both cell lines and primary cultures of bothprokaryotic and eukaryotic cells.

A sample can be from the environment, such as from a body of water orfrom the soil, or from a food, beverage, or water source, an industrialsource, workplace area, public area, or living area. A sample can be anextract, for example a liquid extract of a soil or food sample. A samplecan be a solution made from washing or soaking, or suspending a swabfrom, articles such as tools, articles of clothing, artifacts, or othermaterials.

A sample can be an unprocessed or a processed sample; processing caninvolve steps that increase the purity, concentration, or accessibilityof components of the sample to facilitate the analysis of the sample. Asnonlimiting examples, processing can include steps that reduce thevolume of a sample, remove or separate components of a sample,solubilize a sample or one or more sample components, or disrupt,modify, expose, release, or isolate components of a sample. Nonlimitingexamples of such procedures are centrifugation, precipitation,filtration, homogenization, cell lysis, binding of antibodies, cellseparation, etc.

For example, in some preferred embodiments of the present invention, thesample is a blood sample that is at least partially processed, forexample, by the removal of red blood cells, by concentration, byselection of one or more cell or virus types (for example, white bloodcells or pathogenic cells), or by lysis of cells, etc.

Exemplary samples include a solution of at least partially purifiednucleic acid molecules. The nucleic acid molecules can be from a singlesource or multiple sources, and can comprise DNA, RNA, or both. Forexample, a solution of nucleic acid molecules can be a sample that wassubjected to any of the steps of cell lysis, concentration, extraction,precipitation, nucleic acid selection (such as, for example, poly A RNAselection or selection of DNA sequences comprising Alu elements), ortreatment with one or more enzymes. The sample can also be a solutionthat comprises synthetic nucleic acid molecules.

An oligomer or solid support of the present invention can be anyoligomer format disclosed herein, or any oligomer comprising a monomer,dimer or non nucleic acid component (e.g., linker, fluorophore,quencher, stabilizing moiety) disclosed herein. An oligonucleotideanalogue used in the methods of the present invention can be of anylength and of any nucleobase composition, and can comprise one or morenucleic acid moieties, peptides, proteins lipids, carbohydrates,steroids, and other biochemical and chemical moieties. Anoligonucleotide analogue of the present invention can be provided insolution or bound to a solid support. In some preferred embodiments ofthe present invention, the oligomer comprise non-standard nucleotideanalogues.

Detection methods for bound nucleic acids are well known in the art, andcan include the use of a detectable label that is attached to orincorporated into nucleic acid molecules of the survey population orthat becomes bound to or incorporated into a hybridized target nucleicacid molecule or hybridized target nucleic acid molecule complex.Detectable labels for nucleic acid molecules are well-known in the art,and comprise fluorescent molecules such as fluorophores (including thoseset forth herein), radioisotopes, mass-altered chemical groups, specificbinding members such as biotin that can be detected by signal-generatingmolecules, and the like. Detectable labels can also be incorporated intoor attached to oligomer of the present invention, for example, in caseswhere sandwich hybridization using a signal oligomer is used fordetection, or detection is performed using a specific binding membersuch as an antibody that recognizes oligomer/target nucleic acidmolecule complexes. Solid supports can be scanned, exposed to film,visually inspected, etc. to determine the presence of a detectable labeland thereby determine the binding of a target nucleic acid molecule toan oligomer immobilized on a solid support such as those of theinvention.

Kits

One aspect of the instant invention is the formulation of kits thatfacilitate the practice of syntheses using the compounds of theinvention (such as solid supports of the invention or monomers of theinvention) and assays using oligomers of the invention, as describedabove. The kits of the invention typically comprise a compound of theinvention (such as a solid support of the invention or an oligomer ofthe invention), either present as a chemically reactive species usefulfor preparing conjugates, or present as a completed oligomer where theoligomer is a specific binding pair member. The kit optionally furthercomprises one or more buffering agents, typically present as an aqueoussolution. The kits of the invention optionally further compriseadditional detection reagents, a purification medium for purifying theresulting labeled substance, luminescence standards, enzymes, enzymeinhibitors, organic solvent, or instructions for carrying out an assayof the invention. Other formats for kits will be apparent to those ofskill in the art and are within the scope of the present invention.

By way of summary, in exemplary embodiments, the present inventionprovides:

A nucleic acid probe comprising an oligonucleotide having the structure:5′-Y¹-L¹-L^(Q1)-L²-Y²-3′.Y¹ comprises a sequence of two or more DNA or RNA nucleotides. Y²comprises a sequence of two or more DNA or RNA nucleotides. One of Y¹and Y² has a fluorophore covalently attached (directly or through alinker) to its nucleotide sequence, and the other of Y¹ and Y² has asecond quencher covalently attached (directly or through a linker) toits nucleotide sequence. L¹ and L² are independently selected from abond, substituted or unsubstituted alkyl, substituted or unsubstitutedcycloalkyl, substituted or unsubstituted heteroalkyl, and substituted orunsubstituted heterocycloalkyl. L^(Q1) is

Q¹ is a first quencher having a structure selected from

R^(a), R^(b), and R^(c) are independently selected from substituted orunsubstituted aryl. L³ is selected from a bond, substituted orunsubstituted alkyl, substituted or unsubstituted cycloalkyl,substituted or unsubstituted heteroalkyl, and substituted orunsubstituted heterocycloalkyl.

A nucleic acid probe according to the preceding paragraph, wherein thenucleotide sequence of Y¹ includes: a first nucleotide N¹ having a 5′phosphate covalently attached (directly or through a linker) to thefluorophore or the second quencher, and a second nucleotide N² having a3′ phosphate covalently attached to L¹; and the nucleotide sequence ofY² includes: a third nucleotide N³ having a 5′ phosphate covalentlyattached to L², and a fourth nucleotide N⁴ having a 3′ phosphatecovalently attached (directly or through a linker) to the secondquencher or the fluorophore; wherein when the 5′ phosphate of the firstnucleotide N¹ is covalently attached (directly or through a linker) tothe fluorophore, the 3′ phosphate of the fourth nucleotide N⁴ iscovalently attached (directly or through a linker) to the secondquencher; and when the 5′ phosphate of the first nucleotide N¹ iscovalently attached (directly or through a linker) to the secondquencher, the 3′ phosphate of the fourth nucleotide N⁴ is covalentlyattached (directly or through a linker) to the fluorophore.

A nucleic acid probe according to any preceding paragraph, furthercomprising one or more DNA or RNA nucleotides attached to the linkerthat connects the 5′ phosphate of the first nucleotide N¹ to thefluorophore or second quencher.

A nucleic acid probe according to any preceding paragraph, furthercomprising one or more DNA or RNA nucleotides attached to the linkerthat connects the 3′ phosphate of the fourth nucleotide N⁴ to the secondquencher or fluorophore.

A nucleic acid probe according to any preceding paragraph, wherein the5′ phosphate of the first nucleotide N¹ is covalently attached (directlyor through a linker) to the fluorophore; and the 3′ phosphate of thefourth nucleotide N⁴ is covalently attached (directly or through alinker) to the second quencher.

A nucleic acid probe according to any preceding paragraph, wherein Y¹ isa sequence of 8, 9, 10, or 11 nucleotides.

A nucleic acid probe according to any preceding paragraph, wherein Q¹has a structure selected from:

wherein R^(a2), R^(a3), R^(a5), R^(a6), R^(a7), R^(a8), R^(b2), R^(b3),R^(b4), R^(b5), R^(b6), R^(b7), R^(b8), R^(c2), R^(c3), R^(c4), R^(c5),R^(c6), R^(c7), and R^(c8) are independently selected from H,substituted or unsubstituted alkyl, substituted or unsubstituted alkoxy,and nitro.

A nucleic acid probe according to any preceding paragraph, whereinR^(a2), R^(a3), R^(a5), R^(a6), R^(a7), R^(a8), R^(b2), R^(b3), R^(b4),R^(b5), R^(b6), R^(b7), R^(b8), R^(c2), R^(c3), R^(c4), R^(c5), R^(c6),R^(c7), and R^(c8) are independently selected from H, methyl, ethyl,n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, andnitro.

A nucleic acid probe according to any preceding paragraph, wherein Q¹has the structure:

wherein R^(a2), R^(a3), R^(a5), and R^(a6) are H; at least one ofR^(b2), R^(b3), R^(b5), and R^(b6) is independently methyl or ethyl orn-propyl or isopropyl or n-butyl or isobutyl or sec-butyl or tert-butyl,and the remainder of R^(b2), R^(b3), R^(b5), and R^(b6) are H; and atleast one of R^(c2), R^(c3), R^(c4), R^(c5), and R^(c6) is independentlymethyl or ethyl or n-propyl or isopropyl or n-butyl or isobutyl orsec-butyl or tert-butyl, and the remainder of R^(c2), R^(c3), R^(c4),R^(c5), and R^(c6) are H.

A nucleic acid probe according to any preceding paragraph, wherein theoligonucleotide comprises from 15 to 45 nucleotides.

A nucleic acid probe according to any preceding paragraph, wherein theoligonucleotide comprises from 20 to 30 nucleotides.

A nucleic acid probe according to any preceding paragraph, wherein theoligonucleotide has from 15 to 45 nucleotides.

A nucleic acid probe according to any preceding paragraph, wherein theoligonucleotide has from 20 to 30 nucleotides.

A nucleic acid probe according to any preceding paragraph, wherein L¹and L² are each unsubstituted C₁-C₇ alkyl.

A nucleic acid probe according to any preceding paragraph, wherein L¹and L² are each unsubstituted C₁-C₃ alkyl; and L¹ and L² are the same.

A nucleic acid probe according to any preceding paragraph, wherein L¹and L² are each ethyl.

A nucleic acid probe according to any preceding paragraph, wherein thesecond quencher has the structure:

wherein R^(e2), R^(e5), R^(f2), and R^(f4) are independently selectedfrom H, substituted or unsubstituted alkyl, substituted or unsubstitutedalkoxy, and nitro; and L⁴ is selected from a bond, substituted orunsubstituted alkyl, substituted or unsubstituted cycloalkyl,substituted or unsubstituted heteroalkyl, and substituted orunsubstituted heterocycloalkyl.

A nucleic acid probe according to any preceding paragraph, whereinR^(e2) is unsubstituted C₁-C₄ alkyl; R^(e5) is H; R^(f2) isunsubstituted C₁-C₄ alkyl; and R^(f4) is H.

A nucleic acid probe according to any preceding paragraph, whereinR^(e2) is unsubstituted alkoxy; R^(e5) is unsubstituted C₁-C₄ alkyl;R^(f2) is nitro; and R^(f4) is unsubstituted C₁-C₄ alkyl.

A nucleic acid probe according to any preceding paragraph, whereinR^(e2) is unsubstituted C₁-C₄ alkoxy; R^(e5) is unsubstituted C₁-C₄alkoxy; R^(f2) is H; and R^(f4) is nitro.

A nucleic acid probe according to any preceding paragraph, wherein thesecond quencher has the structure:

wherein L⁴ is selected from a bond, substituted or unsubstituted alkyl,substituted or unsubstituted cycloalkyl, substituted or unsubstitutedheteroalkyl, and substituted or unsubstituted heterocycloalkyl.

A nucleic acid probe according to any preceding paragraph, wherein thesecond quencher is derived from BBQ-650®, Dabcyl, Dabsyl, Eclipse®quencher, Iowa Black® FQ, Iowa Black® RQ-n1, or Iowa Black® RQ-n2.

A nucleic acid probe according to any preceding paragraph, wherein thefluorophore is derived from 6-carboxyfluorescein (FAM),2′7′-dimethoxy-4′5′-dichloro-6-carboxyfluorescein (JOE),tetrachlorofluorescein (TET), 6-carboxyrhodamine (R6G),N,N,N′,N′-tetramethyl-6-carboxyrhodamine (TAMRA), 6-carboxy-X-rhodamine(ROX), 1-dimethylaminonaphthyl-5-sulfonate, 1-anilino-8-naphthalenesulfonate, 2-p-toluidinyl-6-naphthalene sulfonate,5-(2′-aminoethyl)aminonaphthalene-1-sulfonic acid (EDANS), a coumarindye, an acridine dye, indodicarbocyanine 3 (Cy3), indodicarbocyanine 5(Cy5), indodicarbocyanine 5.5 (Cy5.5),3-(1-carboxy-pentyl)-3′-ethyl-5,5′-dimethyloxacarbocyanine (CyA),1H,5H,11H,15H-Xantheno[2,3,4-ij:5,6,7-i′j′]diquinolizin-18-ium, 9-[2(or4)-[[[6-[2,5-dioxo-1-pyrrolidinyl)oxy]-6-oxohexyl]amino]sulfonyl]-4(or2)-sulfophenyl]-2,3,6,7,12,13,16,17-octahydro-inner salt (TR or TexasRed), a BODIPY dye, benzoxaazole, stilbene, or pyrene.

A nucleic acid probe according to any preceding paragraph, wherein thefluorophore is derived from 6-carboxyfluorescein (FAM).

A nucleic acid probe according to any preceding paragraph, wherein thefluorophore has the structure:

wherein L⁵ is selected from a bond, substituted or unsubstituted alkyl,substituted or unsubstituted cycloalkyl, substituted or unsubstitutedheteroalkyl, and substituted or unsubstituted heterocycloalkyl.

A nucleic acid probe according to any preceding paragraph, wherein thenucleotide sequence of Y¹ includes: a first nucleotide N¹ having a 5′phosphate covalently attached (directly or through a linker) to thefluorophore, and a second nucleotide N² having a 3′ phosphate covalentlyattached to L¹; and the nucleotide sequence of Y² includes: a thirdnucleotide N³ having a 5′ phosphate covalently attached to L², and afourth nucleotide N⁴ having a 3′ phosphate covalently attached (directlyor through a linker) to the second quencher; L¹ is unsubstituted C₂alkyl; L² is unsubstituted C₂ alkyl; Q¹ has the structure:

the second quencher has the structure:

wherein L⁴ is selected from a bond, substituted or unsubstituted alkyl,substituted or unsubstituted cycloalkyl, substituted or unsubstitutedheteroalkyl, and substituted or unsubstituted heterocycloalkyl; and thefluorophore comprises a fluorescein moiety.

A nucleic acid probe according to any preceding paragraph, wherein thesecond quencher has the structure:

A nucleic acid probe according to any preceding paragraph, wherein theoligonucleotide comprises from 15 to 45 nucleotides.

A nucleic acid probe according to any preceding paragraph, wherein theoligonucleotide has from 15 to 45 nucleotides.

A nucleic acid probe according to any preceding paragraph, wherein Y¹ isa sequence of from 8 to 11 nucleotides.

A nucleic acid probe according to any preceding paragraph, wherein Y² isa sequence of from 6 to 24 nucleotides.

A nucleic acid probe according to any preceding paragraph, wherein thefluorophore has the structure:

wherein L⁵ is selected from a bond, substituted or unsubstituted alkyl,substituted or unsubstituted cycloalkyl, substituted or unsubstitutedheteroalkyl, and substituted or unsubstituted heterocycloalkyl.

A nucleic acid probe according to any preceding paragraph, wherein thefluorophore has the structure:

A nucleic acid probe according to any preceding paragraph, wherein Y¹ isa sequence of from 8 to 11 nucleotides; Y² is a sequence of from 6 to 24nucleotides; the second quencher has the structure:

andthe fluorophore has the structure:

A nucleic acid probe according to any preceding paragraph, wherein theoligonucleotide is a first oligonucleotide, and the firstoligonucleotide is adapted to hybridize to a second oligonucleotidehaving the structure 3′-Y³-Y⁴-5′, wherein: Y³ comprises a sequence offour or more DNA or RNA nucleotides, including a fifth nucleotide N⁵;and Y⁴ comprises a sequence of four or more DNA or RNA nucleotides,including a sixth nucleotide N⁶ that is directly attached to nucleotideN⁵; wherein if the first oligonucleotide hybridizes to the secondoligonucleotide, N² base pairs with N⁵ and N³ base pairs with N⁶ to forma duplex having a T_(m) that is higher than the T_(m) of a duplex formedbetween the second oligonuleotide and a third oligonucleotide having thestructure 5′-Y¹-Y²-3′.

A nucleic acid probe according to any preceding paragraph, wherein theoligonucleotide is a first oligonucleotide, and the firstoligonucleotide is adapted to hybridize to a second oligonucleotidehaving the structure 3′-Y³-Y⁴-5′, wherein: Y³ comprises a sequence offour or more DNA or RNA nucleotides, including a fifth nucleotide N⁵;and Y⁴ comprises a sequence of four or more DNA or RNA nucleotides,including a sixth nucleotide N⁶ that is directly attached to nucleotideN⁵; wherein if the first oligonucleotide hybridizes to the secondoligonucleotide, N² base pairs with N⁵ and N³ base pairs with N⁶ to forma duplex having a T_(m) that is lower than the T_(m) of a duplex formedbetween the second oligonuleotide and a third oligonucleotide having thestructure 5′-Y¹-Y²-3′.

A method of detecting a second oligonucleotide in a sample, comprising:contacting the sample with a nucleic acid probe according to anypreceding paragraph, wherein the fluorescence of the fluorophore isreduced by donor-acceptor energy transfer to one or both of the firstquencher and the second quencher when the first oligonucleotide is nothybridized to the second oligonucleotide; and detecting an increase influorescence indicating the presence of the second oligonucleotide inthe sample.

A method according to the preceding paragraph, wherein fluorescence isreduced by fluorescence resonance energy transfer when the firstoligonucleotide is not hybridized to the second oligonucleotide.

A method according to any preceding paragraph, wherein fluorescence isreduced by ground state quenching when the first oligonucleotide is nothybridized to the second oligonucleotide.

A method according to any preceding paragraph, wherein fluorescence isreduced by both fluorescence resonance energy transfer and ground statequenching when the first oligonucleotide is not hybridized to the secondoligonucleotide.

A method according to any preceding paragraph, wherein the increase influorescence arises from cleavage of the fluorophore from the firstoligonucleotide.

A method according to any preceding paragraph, wherein the firstoligonucleotide forms a random-coil conformation when the firstoligonucleotide is unhybridized, such that the fluorescence of thefluorophore is reduced.

A method according to any preceding paragraph, wherein the firstoligonucleotide comprises a self-complimentary sequence and wherein thequencher and the fluorophore are attached to the first oligonucleotidesuch that the fluorescence of the fluorophore is quenched by thequencher when the nucleic acid polymer undergoes intramoleeular basepairing.

A method according to any preceding paragraph, wherein the method isused in a PCR reaction wherein synthesis of PCR product results in anincrease in fluorescence.

A method of detecting a second oligonucleotide in a sample, comprising:contacting the sample with a nucleic acid probe according to anypreceding paragraph, wherein the oligonucleotide is a firstoligonucleotide, and the first oligonucleotide is adapted to hybridizeto a second oligonucleotide, and fluorescence of the fluorophore isreduced by donor-acceptor energy transfer to one or both of the firstquencher and the second quencher when the first oligonucleotide is nothybridized to the second oligonucleotide; and detecting an increase influorescence indicating the presence of the second oligonucleotide inthe sample.

A method according to the preceding paragraph, wherein fluorescence isreduced by fluorescence resonance energy transfer when the firstoligonucleotide is not hybridized to the second oligonueleotide.

A method according to any preceding paragraph, wherein fluorescence isreduced by ground state quenching when the first oligonucleotide is nothybridized to the second oligonucleotide.

A method according to any preceding paragraph, wherein fluorescence isreduced by both fluorescence resonance energy transfer and ground statequenching when the first oligonucleotide is not hybridized to the secondoligonucleotide.

A method according to any preceding paragraph, wherein the increase influorescence arises from cleavage of the fluorophore from the firstoligonucleotide.

A method according to any preceding paragraph, wherein the firstoligonucleotide forms a random-coil conformation when the firstoligonucleotide is unhybridized, such that the fluorescence of thefluorophore is reduced.

A method according to any preceding paragraph, wherein the firstoligonucleotide comprises a self-complimentary sequence and wherein thequenchers and the fluorophore are attached to the first oligonucleotidesuch that the fluorescence of the fluorophore is quenched by one or bothof the first quencher and the second quencher when the nucleic acidpolymer undergoes intramoleeular base pairing.

A method according to any preceding paragraph, wherein the method isused in a PCR reaction wherein synthesis of PCR product results in anincrease in fluorescence.

A nucleic acid probe comprising an oligonucleotide having attachedthereto: a first quencher having a structure comprising at least threeresidues selected from substituted or unsubstituted aryl, substituted orunsubstituted heteroaryl, and combinations thereof, wherein at least twoof the residues are covalently linked via an exocyclic diazo bond; and asecond quencher; wherein the first quencher is attached to a nucleotide,which is 8, 9, 10, or 11 nucleotides towards the 3′-terminus from the5′-terminus of the oligonucleotide; and the first quencher and thesecond quencher are attached to the oligonucleotide through a memberindependently selected from a bond, substituted or unsubstituted alkyl,substituted or unsubstituted cycloalkyl, substituted or unsubstitutedheteroalkyl, and substituted or unsubstituted heterocycloalkyl.

A nucleic acid probe according to the preceding paragraph, wherein theoligonucleotide further comprises a fluorophore attached to the5′-terminus of the oligonucleotide through a member selected from abond, substituted or unsubstituted alkyl, substituted or unsubstitutedcycloalkyl, substituted or unsubstituted heteroalkyl, and substituted orunsubstituted heterocycloalkyl.

A nucleic acid probe according to any preceding paragraph, wherein eachof the at least three residues is substituted or unsubstituted phenyl.

A nucleic acid probe according to any preceding paragraph, wherein atleast two of the at least three residues are phenyl substituted with atleast one C₁-C₆ unsubstituted alkyl moiety.

A nucleic acid probe according to any preceding paragraph, wherein thefirst quencher has the structure:

wherein L³ is a member selected from a bond, substituted orunsubstituted alkyl, substituted or unsubstituted cycloalkyl,substituted or unsubstituted heteroalkyl, and substituted orunsubstituted heterocycloalkyl.

A nucleic acid probe according to any preceding paragraph, wherein thesecond quencher has a structure comprising at least three residuesselected from substituted or unsubstituted aryl, substituted orunsubstituted heteroaryl, and combinations thereof, wherein at least twoof the residues are covalently linked via an exocyclic diazo bond.

A nucleic acid probe according to any preceding paragraph, wherein thesecond quencher is attached to the 3′-terminus of the oligonucleotide.

A nucleic acid probe according to any preceding paragraph, wherein thesecond quencher has the structure:

wherein R^(e2), R^(e5), R^(f2), and R^(f4) are independently selectedfrom H, substituted or unsubstituted alkyl, substituted or unsubstitutedalkoxy, and nitro; and L⁴ is selected from a bond, substituted orunsubstituted alkyl, substituted or unsubstituted cycloalkyl,substituted or unsubstituted heteroalkyl, and substituted orunsubstituted heterocycloalkyl.

A nucleic acid probe according to any preceding paragraph, whereinR^(e2) is unsubstituted C₁-C₄ alkyl; R^(e5) is H; R^(f2) isunsubstituted C₁-C₄ alkyl; and R^(f4) is H.

A nucleic acid probe according to any preceding paragraph, whereinR^(e2) is unsubstituted C₁-C₄ alkoxy; R^(e5) is unsubstituted C₁-C₄alkyl; R^(f2) is nitro; and R^(f4) is unsubstituted C₁-C₄ alkyl.

A nucleic acid probe according to any preceding paragraph, whereinR^(e2) is unsubstituted C₁-C₄ alkoxy; R^(e5) is unsubstituted C₁-C₄alkoxy; R² is H; and R^(f4) is nitro.

A nucleic acid probe according to any preceding paragraph, wherein thesecond quencher has the structure:

wherein L⁴ is selected from a bond, substituted or unsubstituted alkyl,substituted or unsubstituted cycloalkyl, substituted or unsubstitutedheteroalkyl, and substituted or unsubstituted heterocycloalkyl.

A nucleic acid probe according to any preceding paragraph, wherein thefluorophore has the structure:

wherein L⁵ is selected from a bond, substituted or unsubstituted alkyl,substituted or unsubstituted cycloalkyl, substituted or unsubstitutedheteroalkyl, and substituted or unsubstituted heterocycloalkyl.

A nucleic acid probe according to any preceding paragraph, wherein thefluorophore has the structure:

A method for detecting a nucleic acid target sequence, the methodcomprising: (a) contacting the target sequence with a detector nucleicacid comprising a single-stranded target binding sequence, wherein thedetector nucleic acid is a nucleic acid probe according to any precedingparagraph, and the detector nucleic acid is in a conformation allowingdonor-acceptor energy transfer between the fluorophore and one or bothof the first quencher and the second quencher when the fluorophore isexcited; (b) hybridizing the target binding sequence to the targetsequence, thereby altering the conformation of the detector nucleicacid, causing a change in a fluorescence parameter; and (c) detectingthe change in the fluorescence parameter, thereby detecting the nucleicacid target sequence.

A method of ascertaining whether a first nucleic acid and a secondnucleic acid hybridize, wherein the first nucleic acid is a nucleic acidprobe according to any preceding paragraph, the method comprising: (a)contacting the first nucleic acid with the second nucleic acid; and (b)detecting an alteration in a fluorescent property of a member selectedfrom the first nucleic acid, the second nucleic acid and a combinationthereof, thereby ascertaining whether the hybridization occurs.

A method of monitoring a nucleic acid amplification reaction, the methodcomprising: (a) preparing an amplification reaction mixture comprising adetector nucleic acid, wherein the detector nucleic acid is a nucleicacid probe according to any preceding paragraph; (b) subjecting theamplification reaction mixture to amplification conditions; (c)monitoring the reaction mixture for a fluorescent signal from thedetector nucleic acid to obtain an assay result; and (d) employing theassay result to monitor the nucleic acid amplification reaction.

A method of detecting amplification of a target sequence, the methodcomprising: (a) hybridizing a sample nucleic acid comprising the targetsequence with PCR primers that flank the target sequence; (b) extendingthe hybridized primers with a polymerase to produce the PCR product, andseparating the two strands of the PCR product to make accessible thesense and antisense strands of the target sequence; (c) hybridizing adetector nucleic acid to the sense or antisense strand of the targetsequence in the PCR product, wherein the detector nucleic acid is anucleic acid probe according to any preceding paragraph; wherein priorto its hybridization to the target sequence, the detector nucleic acidis in a conformation allowing donor-acceptor energy transfer between thefluorophore and one or both of the first quencher and the secondquencher when the fluorophore is excited; thereby altering theconformation of the detector nucleic acid, causing a change in afluorescence parameter; and (d) measuring the change in the fluorescenceparameter to detect the target sequence and its amplification.

The materials and methods of the present invention are furtherillustrated by the examples which follow. These examples are offered toillustrate, but not to limit the claimed invention.

EXAMPLES

Structure

Internal-quenched probes continue to require a 3′ blocker and so asensible approach to improve quenching efficiency is to preserve theterminal quencher for its blocking function while incorporating anadditional quencher at an internal position. FAM-labeled probes werefound to perform most optimally with an internal BHQ0 moiety, which wassurprising since this dye pairing has minimal spectral overlap. Aninternal BHQ0 can be incorporated without regard to base composition bypositioning it between adjacent residues using linkage chemistry thatdisrupts the sugar-phosphate backbone. The phosphoramidite precursorused for incorporation is catalog number BNS-5050 (BiosearchTechnologies, Inc.) with the following chemical structure:

Incorporating the reactive precursor into an oligo during synthesisculminates in an internal BHQ0 modification:

The internal BHQ0 is complemented by an additional BHQ1 moiety on the 3′terminus. The complete structure of this dual quencher FAM-labeled probeis shown in FIG. 3.

The general dye orientation within a dual quencher probe can berepresented as a linear sequence: 5′-[FAM]-oligosequence¹-[I-BHQ0]-oligo sequence²-[BHQ1]-3′

Positioning the internal BHQ0 between residues 9 and 10 was found to beoptimal. Any closer or further removed from the 5′ terminus resulted indiminished signal release. The signal amplification depends not onlyupon sequence position but also quencher-type. Other quenchers likeDABSYL did not work as well, nor did switching the BHQ0/BHQ1 dyeorientation. Even a subtle change in the linkage between the BHQ0chromophore and the oligo is sufficient to disrupt the signal release.Empirical testing revealed an exquisitely sensitive system ofmodifications, and a synergy that is quite unexpected. All of thesedependencies are illustrated in the subsequent experimental section.

Function

The combination of BHQ0 and BHQ1 modifications produces both betterquenching efficiency and signal release, irrespective of oligo length.As shown in FIGS. 4A-C, the same three probe sequences tested previouslywith a T-BHQ1 modification (cf. FIGS. 2A-C) have markedly improvedperformance as dual quencher probes:

FIGS. 4A-C. Amplification traces signaled with an end-labeled probe areshown as slashed lines. Amplification traces signaled with a dualquencher probe are shown as solid lines. The improvement in quenchingefficiency is most pronounced with longer probe sequences, while allthree demonstrate improved signal release. The probe sequences beingcompared are:

21-base probe (end-labeled): (SEQ ID NO: 1)5′-[FAM]-TCCTTTGGGCTCCTGCCATCT-[BHQ1]-3′ 21-base probe (I-BHQ0 +3-BHQ1): (SEQ ID NO: 2)5′-[FAM]-TCCTTTGGG[I-BHQ0]CTCCTGCCATCT[3-BHQ1]-3′29-base probe (end-labeled): (SEQ ID NO: 3)5′-[FAM]-AAACCACTTTATGAAAATCTAACTGGACA-[BHQ1]-3′ 29-base probe (I-BHQ0 +3-BHQ1): (SEQ ID NO: 4) 5′-[FAM]-AAACCACTT[I-BHQ0]TATGAAAATCTAACTGGACA-[3-BHQ1]-3′ 35-base probe (end-labeled): (SEQ ID NO: 5)5′-[FAM]-AAGAGTAGTAGCCTAAGAGTGTCAGTTGTACATCA- [BHQ1]-3′35-base probe (I-BHQ0 + 3-BHQ1): (SEQ ID NO: 6)5′-[FAM]-AAGAGTAGT[I-BHQ0]AGCCTAAGAGTGTCAGTTGTACA TCA-[3-BHQ1]-3′

The improved quenching efficiency can be predicted by the distancedependence of FRET but the improved signal is not easily explained. Onepossibility is the internal quencher must actually have poor spectraloverlap with the fluorescent reporter to better signal theamplification. With a maximal absorbance at 495 nm, the absorptionspectrum of BHQ0 is a bit removed from FAM with its maximal emission at520. Although still speculative, it is possible the internal quenchermust have a lambda max at a shorter wavelength than the reporter, orelse shorter wavelength than the quencher upon the 3′ terminus (BHQ1)for optimal performance characteristics.

It is also possible the two quenchers physically associate with oneanother to produce a complex that interacts more effectively with FAMthan either quencher acting individually. If true, this would produce astructured probe conformation in which the 3′ BHQ1 is brought intocloser proximity to the 5′ fluorescent reporter, and so could beconsidered a conformation-assisted probe.

The improved signal release may originate from favorable interactionsbetween the BHQ0 and Taq polymerase. While traditional end-labeledprobes release signal immediately upon hybridization, it was shown thatin dual quencher probes this signaling mechanism is actually quiteminimal. Even upon hybridization the FRET quenching remains significantdue to the proximity of the fluorescent reporter and internal quencher.Only upon nuclease digestion by the polymerase is the fluorophorecleaved from the quencher-labeled oligo, to release the fluorescentsignal. These sort of enzyme dependencies are very difficult to predict.They may involve subtle electrostatic and hydrophobic interactions withthe protein itself, and difficult to model without X-raycrystallography.

While various hypotheses can explain the improved signal amplification,duplex stabilization is not currently one of them. It was demonstratedthe internal BHQ does not improve binding strength but rather reducesthe probe's T_(M) by 1° C. on average compared to traditionalend-labeled probes. This destabilization might originate from theinternal modification disrupting the sugar-phospate backbone,introducing a bulge of sorts upon binding the target sequence. Thesehypotheses and their characterization are detailed more fully in theexperimental section.

Most candidates were tested by amplifying from a single dilution pointof the target sequence, for efficiency of screening. To ensure no lossof sensitivity near the limit of detection, dual quencher probes weretested across a 7-point dilution series, and their amplification arepresented following analysis by the instrument's normalizationalgorithms:

FIGS. 5A-F. Amplification traces signaled with a end-labeled probe areshown as slashed lines. Amplification traces signaled with a dualquencher probe are shown as solid lines.

Experimental

The improved performance of dual quencher probes can be accomplishedthrough a very specific system of modifications dependent uponchromophore structure, linkage to the oligo, orientation toward oneanother, and location within the sequence. Deviation from any of theseprecise characteristics, however slight, produces inferior performancewith respect to the magnitude of signal release, such as observed withthe probe modified using a T-linked BHQ1.

The chemical structure of this I-TBHQ1 modification is:

That probe contained just a single quencher at an internal positionwhile blocked with a Spacer-C3 modification upon the 3′ terminus:

(SEQ ID NO: 7) 5′-[FAM]-TCCTTTGGGC[I-TBHQ1]CCTGCCATCT-[3-Sp3]-3′

As presented in FIGS. 2A-C, the quenching efficiency was improvedslightly while the signal release was suppressed, with overallperformance compromised as a consequence. Next, probes with a secondBHQ-1 replacing the 3′ Spacer-C3 were tested, as well as replacing theinternal T-BHQ1 with an abasic formulation that disrupts thesugar-phosphate backbone:

FIGS. 6A-B. Amplification traces signaled with an end-labeled probe areshown as slashed lines. Amplification traces signaled with a double BHQ1probe are shown as solid lines. Whether the T-linked formulation in FIG.6A or the abasic formulation in FIG. 6B, both configurations with binaryBHQ1 quenchers have a reduced signal response and compromisedperformance overall. The probe sequences being compared are:

21-base probe (end-labeled): (SEQ ID NO: 1)5′[FAM]-TCCTTTGGGCTCCTGCCATCT-[BHQ1]-3′ 21-base probe (I-TBHQ1 +3-BHQ1): (SEQ ID NO: 8) 5′[FAM]-TCCTTTGGGC[I-TBHQ1]CCTGCCATCT[3-BHQ1]-3′21-base probe (I-BHQ1 + 3-BHQ1): (SEQ ID NO: 9)5′[FAM]-TCCTTTGGG[I-BHQ1]CTCCTGCCATCT-[3-BHQ1]-3′

The abasic formulation of I-BHQ1+3-BHQ1 was tested in a system that isotherwise identical to the dual quencher probes with the configurationof I-BHQ0+3-BHQ1, and yet its performance is much inferior. Results fromonly one representative assay are shown for brevity, but these twoconfigurations have actually been compared across a panel of >10different sequences with an outcome similar to that presentedhere—certain probe sequences have a much reduced signal response fromI-BHQ1, while I-BHQ0 is more frequently improved.

Other binary quencher configurations perform no better, as well asdifferent modifications entirely. 35-base probes with an internalT-Dabsyl (I-TDAB) as well as a truncated version of BHQ0 containing onlythe first aryl moiety (I-Aniline), respectively, were synthesized,always in combination with the 3-BHQ1.

FIGS. 7A-B. Slashed traces are the amplifications signaled with anend-labeled probe. Solid traces are the dual quencher probeI-BHQ0+3-BHQ1, for comparison. Hollow traces are I-TDAB+3-BHQ1 probe inFIG. 7A, and I-Aniline+3-BHQ1 in FIG. 7B. I-TDAB has improved quenchingefficiency but a reduced signal response. I-Aniline has equivalentsignal response to the traditional end-labeled probe but worse quenchingefficiency, indicating the aniline moiety is not functioning as aquencher. Both probe configurations have inferior performance overallcompared to I-BHQ0+3-BHQ1. The probe sequences being compared are:

35-base probe (end-labeled): (SEQ ID NO: 5)5′-[FAM]-AAGAGTAGTAGCCTAAGAGTGTCAGTTGTACATCA- [BHQ1]-3′35-base probe (I-BHQ0 + 3-BHQ1): (SEQ ID NO: 6)5′-[FAM]-AAGAGTAGT[I-BHQ0]AGCCTAAGAGTGTCAGTTGTACAT CA-[3-BHQ1]-3′35-base probe (I-TDAB + 3-BHQ1): (SEQ ID NO: 10)5′-[FAM]-AAGAGTAG[I-TDAB]AGCCTAAGAGTGTCAGTTGTACATC A-[3-BHQ1]-3′35-base probe (I-Aniline + 3-BHQ1): (SEQ ID NO: 11)5′-[FAM]-AAGAGTAGT[I-Aniline]AGCCTAAGAGTGTCAGTTGT ACATCA-[3-BHQ1]-3′

Even changing the linkage between the internal BHQ0 to the oligo willdisrupt the performance of this unique dual quencher probeconfiguration. A 35-base probe with the BHQ0 tethered to a deoxyribosemoiety (I-dRBHQ0), and another with the BHQ0 attached through a glycollinkage that disrupts the sugar phosphate backbone (I-gBHQ0) weresynthesized. The chemical structures of the reactive precursors forthese two modifications are shown below.

FIGS. 8A-B. Amplification traces shown as slashed lines are signaledwith an end-labeled probe. Solid traces are the dual quencher probeI-BHQ0+3-BHQ1, for comparison. Hollow traces are I-dRBHQ0+3-BHQ1 probein FIG. 8A, and I-gBHQ0+3-BHQ1 in FIG. 8B. These modifications maintainthe same chromophore structure while differing only in their linkagechemistry compared to the exemplary I-BHQ0, and yet both have inferiorsignal response. The probe sequences being compared are:

35-base probe (end-labeled): (SEQ ID NO: 5)5′-[FAM]-AAGAGTAGTAGCCTAAGAGTGTCAGTTGTACATCA- [BHQ1]-3′35-base probe (I-BHQ0 + 3-BHQ1): (SEQ ID NO: 6)5′-[FAM]-AAGAGTAGT[I-BHQ0]AGCCTAAGAGTGTCAGTTGTACAT CA-[3-BHQ1]-3′35-base probe (I-dRBHQ0 + 3-BHQ1): (SEQ ID NO: 12)5′-[FAM]-AAGAGTAGT[I-dRBHQ0]AGCCTAAGAGTGTCAGTTGTAC ATCA-[3-BHQ1]-3′35-base probe (I-gBHQ0 + 3-BHQ1): (SEQ ID NO: 13)5′-[FAM]-AAGAGTAGT[I-gBHQ0]AGCCTAAGAGTGTCAGTTGTACA TCA-[3-BHQ1]-3′

Such functional dependencies are not only limited to the internal BHQ0,but also the BHQ1 quencher positioned upon the 3′ terminus. Todemonstrate the importance of this terminal quencher upon the overallprobe configuration, probes with a Spacer C3 instead of BHQ1, whilepreserving the internal BHQ0, were tested. The reverse orientation ofthe two quenchers, so that BHQ1 is positioned at an internal locationwhile BHQ-0 is upon the 3′ terminus, was also tested:

FIGS. 9A-B. Slashed traces are the amplifications signaled with anend-labeled probe. Solid traces are the dual quencher probeI-BHQ0+3-BHQ1, for comparison. Hollow traces are I-BHQ0+3-Sp3 probe inFIG. 9A, and I-BHQ1+3-BHQ0 in FIG. 9B. Both probe configurations haveinferior signal response compared to I-BHQ0+3-BHQ1, emphasizing theimportant role of BHQ1 within the combination as well as the orientationof the quenchers to one another. The probe sequences being compared are:

35-base probe (end-labeled): (SEQ ID NO: 5)5′-[FAM]-AAGAGTAGTAGCCTAAGAGTGTCAGTTGTACATCA- [BHQ1]-3′35-base probe (I-BHQ0 + 3-BHQ1): (SEQ ID NO: 6)5′-[FAM]-AAGAGTAGT[I-BHQ0]AGCCTAAGAGTGTCAGTTGTACAT CA-[3-BHQ1]-3′35-base probe (I-BHQ0 + 3-Sp3): (SEQ ID NO: 14)5′-[FAM]-AAGAGTAGT[I-BHQ0]AGCCTAAGAGTGTCAGTTGTACAT CA-[3-Sp3]-3′35-base probe (I-BHQ1 + 3-BHQ0): (SEQ ID NO: 15)5′-[FAM]-AAGAGTAGT[I-BHQ1]AGCCTAAGAGTGTCAGTTGTACAT CA-[3-BHQ0]-3′

Switching the quencher orientation has a significant effect onfunctionality, but the sequence location is even more sensitive thanthat. The position of the internal BHQ0 was shifted both closer to andmore distant from the 5′ fluorescent reporter, and the consequencetested:

FIG. 10. While the baseline fluorescence is lowered slightly bypositioning the quencher closer to the 5′ reporter, it represents only anominal improvement upon the quenching efficiency of the 9/10configuration (solid lines), especially compared to the traditionalend-labeled probe (slashed lines). However, the signal release iscompromised when the internal BHQ0 is shifted in either direction fromits exemplary position between residues 9 and 10, and so all otherconfigurations have inferior performance. The probe sequences beingcompared are:

35-base probe (end-labeled): (SEQ ID NO: 5)5′-[FAM]-AAGAGTAGTAGCCTAAGAGTGTCAGTTGTACATCA-  [BHQ1]-3′ 6/7 I-BHQ0 +3-BHQ1: (SEQ ID NO: 16)5′-[FAM]-AAGAGT[I-BHQ0]AGTAGCCTAAGAGTGTCAGTTGTACAT CA-[3-BHQ1]-3′7/8 I-BHQ0 + 3-BHQ1: (SEQ ID NO: 17)5′-[FAM]-AAGAGTA[I-BHQ0]GTAGCCTAAGAGTGTCAGTTGTACAT CA-[3-BHQ1]-3′8/9 I-BHQ0 + 3-BHQ1: (SEQ ID NO: 18)5′-[FAM]-AAGAGTAG[I-BHQ0]TAGCCTAAGAGTGTCAGTTGTACAT CA-[3-BHQ1]-3′9/10 I-BHQ0 + 3-BHQ1: (SEQ ID NO: 6)5′-[FAM]-AAGAGTAGT[I-BHQ0]AGCCTAAGAGTGTCAGTTGTACAT CA-[3-BHQ1]-3′11/12 I-BHQ0 + 3-BHQ1: (SEQ ID NO: 19)5′-[FAM]-AAGAGTAGTAG[I-BHQ0]CCTAAGAGTGTCAGTTGTACAT CA-[3-BHQ1]-3′

Improved quenching efficiency can be readily explained through thedistance dependence of FRET, but the signal differences across thevarious probe configurations is quite unexpected. In an attempt todetermine the mechanism of action, the hybridization thermodynamics werecharacterized by combining each probe with its complementary sequenceand then slowly melting apart across a temperature gradient. This meltcurve records the transition temperature, or T_(M), to reveal theprobe's binding stability. Probes that bind at too low a T_(M), beneaththe annealing temperature of amplification, provide a weak signalresponse. Conversely, any increase to the binding stability should havethe opposite effect. 12 different probe designs were tested in thismanner, always comparing the I-BHQ0+3-BHQ1 configuration to thetraditional end-labeled probe. The only difference between the two isthe internal quencher, and so this comparison reveals any contributionto the binding stability from the I-BHQ0 itself:

The melt curves (FIGS. 11A-L) reveal a slight destabilization of −1.0°C. on average from the I-BHQ0+3-BHQ1 dual quencher probe configurationcompared to the traditional probes. Although the results vary somewhatacross the different probe sequences tested, and select sequences doregister a higher T_(M) for the dual quencher configuration, overallthere is no significant stabilization being contributed by the BHQ0. Assuch, the thermodynamics of hybridization does not appear to be a factorin the signaling mechanism of this dual quencher probe-type.

More interesting is the change in signal between hybridized andun-hybridized conformations at 60° C., as gauged by comparing theslashed and solid traces to their corresponding hollow traces. Thesehollow traces represent each probe in the absence of its complementacross the same range, and at higher temperatures will overlay theslashed and solid traces since all the strands are melted apart. Fromthe melt curves alone, one might conclude the traditional probe is thebest performing: it has much greater signal release upon hybridizationthan the dual quencher probe configuration. And yet in contrast to thishybridization assay, the PCR amplifications produce a very differentresult: the dual quencher probe has an equivalent or greater signalrelease than the traditional probe. The PCR result is surprising sincethe melt curve data at 60° C. reveals that, even upon hybridization totheir complement, the dual quencher probe remains efficientlyquenched—better quenched than all but the shortest traditional probesleft unhybridized. All of this suggests that in PCR assay the dominantsignaling mechanism is hydrolysis rather than hybridization. Probehydrolysis is accomplished through the 5′→3′ exonuclease activity of thepolymerase and so it would appear that dual quencher probes gain potencythrough their interactions with this enzyme. The synergy of this systemof modifications in combination with the polymerase would be verydifficult to anticipate or engineer. It is only accomplished throughsignificant empirical testing, with dozens of different configurationsevaluated for their functionality in the context of qPCR.

Methods

qPCR Reaction Composition Volume Concentration Nuclease-free water 6.4μL N/A 2X Bioline ImmoMix (w/3 mM MgCl₂) 10.0 μL  1X Additional MgCl₂(50 mM) 1.2 μL 6 Mm total Stock Forward Primer (10 μM) 0.6 μL 300 nMStock Reverse Primer (10 μM) 0.6 μL 300 nM Stock Probe (10 μM) 0.6 μL300 nM Sample DNA 1.0 μL variable Total: 20.0 μL  qPCR Thermal CyclingRoutine Enzyme Activation 95° C. for 10 minutes Followed by: 50 cyclesof 95° C. for 20 seconds 60° C. for 60 seconds with signal detected atthe 60° C. annealing temp Melt Curve Reaction Composition 10 mM Tris 50mM NaCl 5 mM MgCl₂ 2 μM Probe 2 μM complement (or water) Melt CurveTemperature Gradient Initial Denaturation 95° C. for 5 minutes followedby: temperature ramp 25° C. to 95° C. rising 0.1° C. at each step, andwaiting for 2 seconds at each step

It is understood that the examples and embodiments described herein arefor illustrative purposes only and that various modifications or changesin light thereof will be suggested to persons skilled in the art and areto included within the spirit and purview of this application and areconsidered within the scope of the appended claims. All publications,patents, and patent applications cited herein are hereby incorporated byreference in their entirety for all purposes.

What is claimed is:
 1. A nucleic acid probe comprising: an oligonucleotide having the structure 5′-Y¹-L¹-L^(Q1)-L²-Y²-3′, wherein Y¹ comprises a sequence of two or more DNA or RNA nucleotides; Y² comprises a sequence of two or more DNA or RNA nucleotides; wherein one of Y¹ and Y² has a fluorophore covalently attached to its nucleotide sequence, and the other of Y¹ and Y² has a second quencher covalently attached to its nucleotide sequence; L¹ and L² are independently selected from a bond, substituted or unsubstituted alkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heteroalkyl, and substituted or unsubstituted heterocycloalkyl; and L^(Q1) is:

wherein R^(b2) and R^(c2) are independently selected alkyl moieties.
 2. The nucleic acid probe of claim 1, wherein the nucleotide sequence of Y¹ includes: a first nucleotide N¹ having a 5′ phosphate covalently attached to said fluorophore or said second quencher, and a second nucleotide N² having a 3′ phosphate covalently attached to L¹; and the nucleotide sequence of Y² includes: a third nucleotide N³ having a 5′ phosphate covalently attached to L², and a fourth nucleotide N⁴ having a 3′ phosphate covalently attached to said second quencher or said fluorophore; wherein when the 5′ phosphate of said first nucleotide N¹ is covalently attached to said fluorophore, the 3′ phosphate of said fourth nucleotide N⁴ is covalently attached (directly or through a linker) to said second quencher; and when the 5′ phosphate of said first nucleotide N¹ is covalently attached to said second quencher, the 3′ phosphate of said fourth nucleotide N⁴ is covalently attached to said fluorophore.
 3. The nucleic acid probe of claim 2, further comprising one or more DNA or RNA nucleotides attached to the linker that connects the 5′ phosphate of said first nucleotide N¹ to said fluorophore or second quencher.
 4. The nucleic acid probe of claim 2, further comprising one or more DNA or RNA nucleotides attached to the linker that connects the 3′ phosphate of said fourth nucleotide N⁴ to said second quencher or fluorophore.
 5. The nucleic acid probe of claim 2, wherein the 5′ phosphate of said first nucleotide N¹ is covalently attached to said fluorophore; and the 3′ phosphate of said fourth nucleotide N⁴ is covalently attached to said second quencher.
 6. The nucleic acid probe of claim 5, wherein Y¹ is a sequence of 8, 9, 10, or 11 nucleotides.
 7. The nucleic acid probe of claim 1, wherein said oligonucleotide comprises from 15 to 45 nucleotides.
 8. The nucleic acid probe of claim 7, wherein said oligonucleotide comprises from 20 to 30 nucleotides.
 9. The nucleic acid probe of claim 1, wherein said oligonucleotide has from 15 to 45 nucleotides.
 10. The nucleic acid probe of claim 9, wherein said oligonucleotide has from 20 to 30 nucleotides.
 11. The nucleic acid probe of claim 1, wherein L¹ and L² are each unsubstituted C₁-C₇ alkyl.
 12. The nucleic acid probe of claim 11, wherein L¹ and L² are each unsubstituted C₁-C₃ alkyl; and L¹ and L² are the same.
 13. The nucleic acid probe of claim 12, wherein L¹ and L² are each ethyl.
 14. The nucleic acid probe of claim 1, wherein the second quencher has the structure:

wherein R^(e2), R^(e5), R^(f2),and R^(f4) are independently selected from H, substituted or unsubstituted alkyl, substituted or unsubstituted alkoxy, and nitro; and L⁴ is selected from a bond, substituted or unsubstituted alkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heteroalkyl, and substituted or unsubstituted heterocycloalkyl.
 15. The nucleic acid probe of claim 14, wherein R^(e2) is unsubstituted C₁-C₄ alkyl; R^(e5) is H; R^(f2) is unsubstituted C₁-C₄ alkyl; and R^(f4) is H.
 16. The nucleic acid probe of claim 14, wherein R^(e2) is unsubstituted C₁-C₄ alkoxy; R^(e5) is unsubstituted C₁-C₄ alkyl; R^(f2) is nitro; and R^(f4) is unsubstituted C₁-C₄ alkyl.
 17. The nucleic acid probe of claim 14, wherein R^(e2) is unsubstituted C₁-C₄ alkoxy; R^(e5) is unsubstituted C₁-C₄ alkoxy; R^(f2) is H; and R^(f4) is nitro.
 18. The nucleic acid probe of claim 1, wherein the second quencher has the structure:

wherein L⁴ is selected from a bond, substituted or unsubstituted alkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heteroalkyl, and substituted or unsubstituted heterocycloalkyl.
 19. The nucleic acid probe of claim 1, wherein the second quencher is derived from BBQ-650®, Dabcyl, Dabsyl, Eclipse® quencher, Iowa Black® FQ, Iowa Black® RQ-n1, or Iowa Black® RQ-n2.
 20. The nucleic acid probe of claim 1, wherein the fluorophore is derived from 6-carboxyfluorescein (FAM), 2′7′-dimethoxy-4′5′-dichloro-6-carboxyfluorescein (JOE), tetrachlorofluorescein (TET), 6-carboxyrhodamine (R6G), N,N,N′,N′-tetramethyl-6-carboxyrhodamine (TAMRA), 6-carboxy-X-rhodamine (ROX), 1-dimethylaminonaphthyl-5-sulfonate, 1-anilino-8-naphthalene sulfonate, 2-p-toluidinyl-6-naphthalene sulfonate, 5-(2′-aminoethyl)aminonaphthalene-1-sulfonic acid (EDANS), a coumarin dye, an acridine dye, indodicarbocyanine 3(Cy3), indodicarbocyanine 5(Cy5), indodicarbocyanine 5.5(Cy5.5), 3-(1-carboxy-pentyl)-3′-ethyl-5,5′-dimethyloxacarbocyanine (CyA), 1H, 5H, 11H, 15H-Xantheno[2,3,4-ij:5,6,7-i′j′]diquinolizin-18-ium, 9-[2(or 4)-[[[6-[2,5-dioxo-1-pyrrolidinyl)oxy]-6-oxohexyl]amino]sulfonyl]-4(or 2)-sulfophenyl]-2,3,6,7,12,13,16,17-octahydro-inner salt (TR or Texas Red), a BODIPY dye, benzoxaazole, stilbene, or pyrene.
 21. The nucleic acid probe of claim 1, wherein the fluorophore is derived from 6-carboxyfluorescein (FAM).
 22. The nucleic acid probe of claim 1, wherein the fluorophore has the structure:

wherein L⁵ is selected from a bond, substituted or unsubstituted alkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heteroalkyl, and substituted or unsubstituted heterocycloalkyl.
 23. The nucleic acid probe of claim 1, wherein the nucleotide sequence of Y¹ includes: a first nucleotide N¹ having a 5′ phosphate covalently attached to said fluorophore, and a second nucleotide N² having a 3′ phosphate covalently attached to L¹; and the nucleotide sequence of Y² includes: a third nucleotide N³ having a 5′ phosphate covalently attached to L², and a fourth nucleotide N⁴ having a 3′ phosphate covalently attached to said second quencher; L¹ is unsubstituted C₂ alkyl; L² is unsubstituted C₂ alkyl; Q¹ has the structure:

said second quencher has the structure:

wherein L⁴ is selected from a bond, substituted or unsubstituted alkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heteroalkyl, and substituted or unsubstituted heterocycloalkyl; and said fluorophore comprises a fluorescein moiety.
 24. The nucleic acid probe of claim 23, wherein the second quencher has the structure:


25. The nucleic acid probe of claim 23, wherein said oligonucleotide comprises from 15 to 45 nucleotides.
 26. The nucleic acid probe of claim 25, wherein said oligonucleotide has from 15 to 45 nucleotides.
 27. The nucleic acid probe of claim 23, wherein Y¹ is a sequence of from 8 to 11 nucleotides.
 28. The nucleic acid probe of claim 23, wherein Y² is a sequence of from 6 to 24 nucleotides.
 29. The nucleic acid probe of claim 23, wherein the fluorophore has the structure:

wherein L⁵ is selected from a bond, substituted or unsubstituted alkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heteroalkyl, and substituted or unsubstituted heterocycloalkyl.
 30. The nucleic acid probe of claim 29, wherein the fluorophore has the structure:


31. The nucleic acid probe of claim 23, wherein Y¹ is a sequence of from 8 to 11 nucleotides; Y² is a sequence of from 6 to 24 nucleotides; the second quencher has the structure:

and the fluorophore has the structure:


32. The nucleic acid probe of claim 2, wherein the oligonucleotide is a first oligonucleotide, and the first oligonucleotide hybridizes to a second oligonucleotide having the structure 3′-Y³-Y⁴-5′, wherein: Y³ comprises a sequence of four or more DNA or RNA nucleotides, including a fifth nucleotide N⁵; and Y⁴ comprises a sequence of four or more DNA or RNA nucleotides, including a sixth nucleotide N⁶ that is directly attached to nucleotide N⁵; wherein if the first oligonucleotide hybridizes to the second oligonucleotide, N² base pairs with N⁵ and N³ base pairs with N⁶ to form a duplex having a T_(m) that is higher than the T_(m) of a duplex formed between the second oligonuleotide and a third oligonucleotide having the structure 5′-Y¹-Y²-3′.
 33. The nucleic acid probe of claim 2, wherein the oligonucleotide is a first oligonucleotide, and the first oligonucleotide hybridizes to a second oligonucleotide having the structure 3′-Y³-Y⁴-5′, wherein: Y³ comprises a sequence of four or more DNA or RNA nucleotides, including a fifth nucleotide N⁵; and Y⁴ comprises a sequence of four or more DNA or RNA nucleotides, including a sixth nucleotide N⁶ that is directly attached to nucleotide N⁵; wherein if the first oligonucleotide hybridizes to the second oligonucleotide, N² base pairs with N⁵ and N³ base pairs with N⁶ to form a duplex having a T_(m) that is lower than the T_(m) of a duplex formed between the second oligonuleotide and a third oligonucleotide having the structure 5′-Y¹-Y²-3′.
 34. A method of detecting a second oligonucleotide in a sample, comprising: contacting the sample with the nucleic acid probe of claim 33, wherein the fluorescence of the fluorophore is reduced by donor-acceptor energy transfer to one or both of the first quencher and the second quencher when the first oligonucleotide is not hybridized to the second oligonucleotide; and detecting an increase in fluorescence indicating the presence of the second oligonucleotide in the sample.
 35. The method of claim 34, wherein fluorescence is reduced by fluorescence resonance energy transfer when the first oligonucleotide is not hybridized to the second oligonucleotide.
 36. The method of claim 34, wherein fluorescence is reduced by ground state quenching when the first oligonucleotide is not hybridized to the second oligonucleotide.
 37. The method of claim 34, wherein fluorescence is reduced by both fluorescence resonance energy transfer and ground state quenching when the first oligonucleotide is not hybridized to the second oligonucleotide.
 38. The method of claim 34, wherein the increase in fluorescence arises from cleavage of the fluorophore from the first oligonucleotide.
 39. The method of claim 34, wherein the first oligonucleotide forms a random-coil conformation when the first oligonucleotide is unhybridized, such that the fluorescence of the fluorophore is reduced.
 40. The method of claim 34, wherein the first oligonucleotide comprises a self-complimentary sequence and wherein the quencher and the fluorophore are attached to the first oligonucleotide such that the fluorescence of the fluorophore is quenched by the quencher when the nucleic acid polymer undergoes intramoleeular base pairing.
 41. The method of claim 34, wherein the method is used in a PCR reaction wherein synthesis of PCR product results in an increase in fluorescence.
 42. A method of detecting a second oligonucleotide in a sample, comprising: contacting the sample with the nucleic acid probe of claim 1, wherein the oligonucleotide is a first oligonucleotide, and the first oligonucleotide is adapted to hybridize to a second oligonucleotide, and fluorescence of the fluorophore is reduced by donor-acceptor energy transfer to one or both of the first quencher and the second quencher when the first oligonucleotide is not hybridized to the second oligonucleotide; and detecting an increase in fluorescence indicating the presence of the second oligonucleotide in the sample.
 43. The method of claim 42, wherein fluorescence is reduced by fluorescence resonance energy transfer when the first oligonucleotide is not hybridized to the second oligonueleotide.
 44. The method of claim 42, wherein fluorescence is reduced by ground state quenching when the first oligonucleotide is not hybridized to the second oligonucleotide.
 45. The method of claim 42, wherein fluorescence is reduced by both fluorescence resonance energy transfer and ground state quenching when the first oligonucleotide is not hybridized to the second oligonucleotide.
 46. The method of claim 42, wherein the increase in fluorescence arises from cleavage of the fluorophore from the first oligonucleotide.
 47. The method of claim 42, wherein the first oligonucleotide forms a random-coil conformation when the first oligonucleotide is unhybridized, such that the fluorescence of the fluorophore is reduced.
 48. The method of claim 42, wherein the first oligonucleotide comprises a self-complimentary sequence and wherein the quenchers and the fluorophore are attached to the first oligonucleotide such that the fluorescence of the fluorophore is quenched by one or both of the first quencher and the second quencher when the nucleic acid polymer undergoes intramoleeular base pairing.
 49. The method of claim 42, wherein the method is used in a PCR reaction wherein synthesis of PCR product results in an increase in fluorescence.
 50. A method for detecting a nucleic acid target sequence, said method comprising: (a) contacting said target sequence with a detector nucleic acid comprising a single-stranded target binding sequence, wherein said detector nucleic acid is a nucleic acid probe according to claim 1, and said detector nucleic acid is in a conformation allowing donor-acceptor energy transfer between said fluorophore and one or both of said first quencher and said second quencher when said fluorophore is excited; (b) hybridizing said target binding sequence to said target sequence, thereby altering said conformation of said detector nucleic acid, causing a change in a fluorescence parameter; and (c) detecting said change in said fluorescence parameter, thereby detecting said nucleic acid target sequence.
 51. A method of ascertaining whether a first nucleic acid and a second nucleic acid hybridize, wherein said first nucleic acid is a nucleic acid probe according to claim 1, said method comprising: (a) contacting said first nucleic acid with said second nucleic acid; and (b) detecting an alteration in a fluorescent property of a member selected from said first nucleic acid, said second nucleic acid and a combination thereof, thereby ascertaining whether said hybridization occurs.
 52. A method of monitoring a nucleic acid amplification reaction, said method comprising: (a) preparing an amplification reaction mixture comprising a detector nucleic acid, wherein said detector nucleic acid is a nucleic acid probe according to claim 1; (b) subjecting the amplification reaction mixture to amplification conditions; (c) monitoring the reaction mixture for a fluorescent signal from the detector nucleic acid to obtain an assay result; and (d) employing the assay result to monitor the nucleic acid amplification reaction.
 53. A method of detecting amplification of a target sequence, said method comprising: (a) hybridizing a sample nucleic acid comprising the target sequence with PCR primers that flank the target sequence; (b) extending the hybridized primers with a polymerase to produce the PCR product, and separating the two strands of the PCR product to make accessible the sense and antisense strands of the target sequence; (c) hybridizing a detector nucleic acid to the sense or antisense strand of the target sequence in the PCR product, wherein the detector nucleic acid is a nucleic acid probe according to claim 1; wherein prior to its hybridization to the target sequence, the detector nucleic acid is in a conformation allowing donor-acceptor energy transfer between the fluorophore and one or both of the first quencher and the second quencher when the fluorophore is excited; thereby altering the conformation of the detector nucleic acid, causing a change in a fluorescence parameter; and (d) measuring the change in the fluorescence parameter to detect the target sequence and its amplification.
 54. A method of detecting a second oligonucleotide in a sample, comprising: contacting the sample with the nucleic acid probe of claim 32, wherein the fluorescence of the fluorophore is reduced by donor-acceptor energy transfer to one or both of the first quencher and the second quencher when the first oligonucleotide is not hybridized to the second oligonucleotide; and detecting an increase in fluorescence indicating the presence of the second oligonucleotide in the sample.
 55. The nucleic acid probe according to claim 1, wherein the oligonucleotide is hybridized to a second oligonucleotide forming a duplex, with a lower T_(m) than an identical duplex without the first quencher.
 56. The nucleic acid probe according to claim 1, wherein Q¹ is located between positions 9 and 10 from the 3′-terminus. 